UNA single stranded oligomers for therapeutics

ABSTRACT

This invention provides UNA single-stranded oligomers for therapeutics. The UNA oligomers can be composed of one or more 2′-3′-seco-nucleomonomers and one or more natural or non-natural nucleotide monomers. Embodiments include UNA oligomers with phosphorothioate or boranophosphate intermonomer linkages. The UNA oligomers can be used for therapeutics that target oligonucleotides, nucleic acids, or RNAs to reduce their activity.

SEQUENCE LISTING

This application includes a sequence listing submitted herewith via EFSas an ASCII file created on Nov. 19, 2009, named ARC854US_SL.txt, whichis 5490 bytes in size, and is hereby incorporated by reference in itsentirety.

BACKGROUND

RNA interference (RNAi) has attracted massive attention in recent years,as it provides a means to silence the expression of a target gene. Itprovides basic research with a method for studying genetic andbiochemical pathways, and the function of individual genes and geneproducts. Consequently, RNA interference has become a critical tool fortarget validation in the pharmaceutical industry. Moreover, substantialinvestments have been made with the goal of developing RNA complexescapable of mediating RNA interference complexes that can be used asdrugs.

The attractiveness of RNAi for use in therapy lies in its sensitivityand sequence specificity. However, concerns have arisen concerningsequence specificity, e.g. because the wrong strand of the RNA complexmay direct the response to the wrong target nucleic acids. Moreover, RNAcomplexes of a certain size induce a non-specific interferon dependentresponse, which is also undesirable.

Patent application US2003/0108923 describes RNA complexes capable ofmediating RNAi comprising an antisense strand and a passenger strand,wherein the strands are 21-23 nucleotides in length. It is suggestedthat the RNA complexes are used for therapeutic applications.

Similarly, patent application US2005/0234007 describes RNA complexescapable of mediating RNAi comprising an antisense strand and a passengerstrand, wherein the complex comprises 3′-overhangs. It is suggested thatthe RNA complexes are used for therapeutic applications.

WO2005/073378 describes RNAi complexes containing chemically modifiednucleotides capable of mediating RNAi comprising an antisense strand anda passenger strand. The RNA complexes described in the specificationcomprise LNA residues and it is stated that incorporation of LNAresidues near the 5′end of one of the strand can control which strand isincorporated in the RISC complex, because the strand that forms theweakest base pair at its 5-end is incorporated into the RISC complex.

RNAi is only one of several strategies for mediating inhibition of geneexpression using oligonucleotides, including the RNA complexes of thisinvention. These different strategies, that include RNase H mediated RNAcleavage, steric block RNA binding, DNAzyme or Ribozyme mediated RNAcleavage and siRNA approaches have been described in the literaturetogether with the nature of selected chemically modified nucleotidesthat are compatible with biological activity [J. Kurreck, Eur. J.Biochem. 2003, 270, 1628].

The hydroxymethyl substituted monomers B-E of the invention have beenincorporated into DNA strands, and therefore procedures for preparationof their phosphoramidite building blocks for automated DNA/RNA synthesishave been reported [K. D. Nielsen et al., Bioorg. Med. Chem. 1995, 3,1493; H. Thrane et al., Tetrahedron 1995, 51, 10389; P. Nielsen et al.,Bioorg. Med. Chem. 1995, 3, 19]. It is exclusively thymine monomers thathave been incorporated into DNA strands. None of the hydroxymethylsubstituted monomers have previously been incorporated into RNA strands.

In one report, one or two 2′-secouridines was incorporated into a DNAoligonucleotide and a positive effect on RNase H mediated RNAdegradation was observed (Mangos M M, Min K L, Viazovkina E, GalarneauA, Elzagheid M I, Parniak M A, Damha M J., J Am Chem Soc. 2003 Jan. 22;125(3):654-61.).

SUMMARY

The present invention provides RNA complexes with one or morehydroxymethyl substituted monomers incorporated into an RNA strand to beused in relation to RNA-guided gene regulation or gene analysis, inparticular RNA interference. Thus, it is an object of the presentinvention to provide RNA complexes, which have reduced off targeteffects as compared to the RNA complexes typically used. Another objectis to provide RNA complexes which induce a reduced interferon response.Still another object is to provide RNA complexes with improvedproperties with regard to stability towards enzymatic degradation incell cultures or in vivo. Still another object is to provide RNAcomplexes that display enhanced gene regulatory function, e.g. genesilencing effect, in cell cultures or in vivo, relative to theunmodified RNA complexes. Yet further objects are to provide RNAcomplexes that are targeted towards specific organs or tissue, and thatare capable of penetrating the cell membrane. The present invention alsoprovides monomers suitable for incorporation of hydroxymethylsubstituted monomers into oligonucleotides and methods for theirsynthesis.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 Examples of the different architectures of the hydroxymethylsubstituted nucleotides that are incorporated in the RNA complexes areshown. Monomer A is shown for comparison and is a natural RNA monomerwith its ribose scaffold. The characteristic of Monomers B-E that arecomprised in the RNA complexes of the invention is that they contain asubstituent that is a hydroxymethyl group (“the free hydroxymethylgroup”), and therefore the invention is entitled “Hydroxymethylsubstituted RNA oligonucleotides and RNA complexes”. The freehydroxymethyl group is for example attached at the C4′ atom of a cyclicribose scaffold or the C1′ atom of an acyclic ribose-based scaffold. Thehydroxymethyl substituted nucleotides of the invention contain otheroxygen atoms that are each attached to a phosphorus atom and thuspartake in the formation of internucleotide linkages (see FIG. 1). Oneor more of these other oxygen atoms can be part of a hydroxy group whichis the case when one or more of the hydroxymethyl substitutednucleotides of the RNA complexes of the invention is (are) positioned atthe 3′- or 5′-end of an RNA strand. When one of the hydroxymethylsubstituted nucleotides of the RNA complexes of the invention ispositioned at the 3′-end and/or the 5′-end of the RNA strands, ahydroxyl group of this monomer can be phosphorylated, as can be the casefor any terminally positioned natural RNA monomer. To the hydroxymethylsubstituted nucleotides of the invention is attached a nucleobase likeuracil, thymine, cytosine, 5-methylcytosine, adenine, guanine or anyother known natural or synthetic nucleobase or nucleobase analogue(designated as “Base” in FIG. 1).

FIG. 2 Derivatised, functionalised and conjugated variants of thehydroxymethyl substituted monomers are shown. As examples are shownderivatised, functionalised and conjugated variants of the hydroxymethylsubstituted 2′,3′-seco-RNA monomer D (see FIG. 1). Monomer F contains agroup R linked via an ether linkage. Monomer G contains a group R linkedvia a thioether linkage. Monomer H contains a group R linked via anamide linkage. Monomer I contains a group R linked via an amino linkage.Monomer J contains a group R linked via a piperazino unit. Byincorporation of one or several of such monomers into the RNA complexesof the invention, the properties of the RNA complexes can be modulated.For example can increased biostability, increased RNA targetingcapability or specific delivery properties be introduced, andfluorescent groups can be attached for detection purposes.

FIG. 3 Structures of two of the hydroxymethyl substituted monomers(Monomer C and Monomer D) that may be a monomer of an oligonucleotide orRNA complex.

FIG. 4 Gene silencing results for siRNA complexes of the inventioncontaining “monomer X” (i.e., 2′,3′-seco-RNA Monomer D). Results wereobtained with the W130 sense strand (see FIG. 4 for nucleotide sequence)containing Monomer D having uracil as the nucleobase (shown as ‘X’ inthe the W130 sense strand). The nucleic acid sequence of the antisensestrands used in this study are listed at the bottom of this figure (allX monomers in the antisense sequences are Monomer D having uracil as thenucleobase). Monomers with a superscript “L” represent a locked nucleicacid (e.g., T^(L) indiciates a thymine locked nucleic acid or LNA).

FIG. 5 Gene silencing results for siRNA complexes of the inventioncontaining “monomer X” (i.e., 2′,3′-seco-RNA Monomer D). Results wereobtained with the W131 sense strand (see FIG. 5 for nucleotide sequence)containing Monomer D having uracil as the nucleobase (shown as ‘X’ inthe the W131 sense strand). The nucleic acid sequence of the antisensestrands used in this study are listed at the bottom of FIG. 4 (all Xmonomers in the antisense sequences are Monomer D having uracil as thenucleobase).

FIG. 6 Gene silencing results for siRNA complexes of the inventioncontaining “monomer X” (i.e., 2′,3′-seco-RNA Monomer D). These resultswere obtained with the W282 sense strand (see FIG. 6 for nucleotidesequence) containing Monomer D having as nucleobase cytosine (sC, firstX from the 5′-end of the W282 sequence), adenine (sA, second X from the5′-end of the W282 sequence) and cytosine (sC, last X from the 3′-end ofthe W282 sequence). The nucleic acid sequence of the antisense strandsused in this study are listed at the bottom of FIG. 4 (all X monomers inthe antisense sequences are Monomer D having uracil as the nucleobase).

FIG. 7 Gene silencing results for siRNA complexes of the inventioncontaining “monomer X” (i.e., 2′,3′-seco-RNA Monomer D). These resultswere obtained with the W194 sense strand (see FIG. 7 for nucleotidesequence). The nucleic acid sequence of the antisense strands used inthis study are listed at the bottom of FIG. 4 (all X monomers in theantisense sequences are Monomer D having uracil as the nucleobase).Monomers with a superscript “L” represent a locked nucleic acid (e.g.,T^(L) indiciates a thymine locked nucleic acid or LNA).

FIG. 8 Gene silencing results for siRNA complexes of the inventioncontaining “monomer X” (i.e., 2′,3′-seco-RNA Monomer D). These resultswere obtained with the W181 sense strand (see FIG. 8 for nucleotidesequence). The nucleic acid sequence of the antisense strands used inthis study are listed at the bottom of FIG. 4 (all X monomers in theantisense sequences are Monomer D having uracil as the nucleobase).Monomers with a superscript “L” represent a locked nucleic acid (e.g.,T^(L) indiciates a thymine locked nucleic acid or LNA).

FIG. 9 Gene silencing results for siRNA complexes of the inventioncontaining “monomer X” (i.e., 2′,3′-seco-RNA Monomer D). These resultswere obtained with the W129 sense strand (see FIG. 9 for nucleotidesequence) containing Monomer D having uracil as the nucleobase (shown as‘X’ in the the W129 sense strand). The antisense strands included inthis study are listed at the bottom of FIG. 4 (all X monomers in theantisense sequences are Monomer D having uracil as the nucleobase).Monomers with a superscript “L” represent a locked nucleic acid (e.g.,T^(L) indiciates a thymine locked nucleic acid or LNA).

DETAILED DESCRIPTION

Specific features described in one aspect of the invention also apply toother aspects of the invention. E.g. features described with regards tothe RNA complexes of the first aspect also apply to the oligonucleotidesof the ninth aspect and to the RNA duplexes of the tenth aspect whereappropriate.

First Aspect, RNA Complexes

RNA complexes in the form of siRNA duplexes or single stranded RNA canmediate various modifications of target nucleic acids in the cell. Inthis process, the antisense strand of the complex acts as a guide, asthe antisense strand can hybridise to target nucleic acids that havestretches of sequence complementarity to the antisense strand.

Before targeting of a target nucleic acid, the antisense strand is oftenincorporated into an RNA guided protein complex (RGPC), which can actupon the target nucleic acid. One example of a RNA guided proteincomplex is the RNA Induced Silencing Complex (RISC). It is believed thatother such RGPCs exist and that the RNA complexes of the presentinvention will also be of advantage, when used with these other RGPCs oreven without interacting with any RGPCs.

One object of the present invention is to stabilise the RNA complexestowards nucleolytic degradation in biological media (serum, in vivo, incell cultures).

Another object of the present invention is to improve the gene silencingeffect of a double stranded RNA complex. This improvement can, e.g.relate to increased potency, reduced off-target effects, reduced immunestimulation, increased stability for storage, increased stability inbiological media like serum etc., increased duration of action andimproved pharmacokinetic properties, all relative to the nativeunmodified RNA complex.

Yet another object of the present invention is to improve the genesilencing effect of a single stranded RNA oligonucleotide. Thisimprovement can, e.g., relate to increased potency, reduced off-targeteffects, reduced immune stimulation, increased stability for storage,increased stability in biological media like serum etc., increasedduration of action and improved pharmacokinetic properties, all relativeto the native unmodified RNA complex.

It is an object of the invention to secure that only the antisensestrand, and not the passenger strand, of an siRNA complex of theinvention will mediate modifications of target nucleic acids. Thefulfilment of this object will provide RNA complexes with less offtarget effects.

Another object of the invention is to ensure sufficient stability of anRNA complex in biological media. Thus it is an object to provide RNAcomplexes that display enhanced gene regulatory function, e.g. genesilencing effect, in cell cultures or in vivo, relative to unmodifiedRNA complexes.

The basic idea of the invention is to incorporate one or morehydroxymethyl substituted monomers into an RNA complex of the invention.In case of siRNA this could lead to preferential incorporation of onlyone strand of the complex into RISC. Incorporation of one or morehydroxymethyl substituted monomers into one (or more) RNA strand(s) ofan RNA complex will improve the life time of the RNA complex inbiological media and in vivo, and thus will lead to improved biologicalactivity, for example improved gene regulation activity.

An RNA strand of an RNA complex of the invention may comprise naturalRNA nucleotides, RNA modifications known to be compatible with genesilencing activity [Nawrot and Sipa, Curr. Topics Med. Chem. 2006, 6,913-925], and the hydroxymethyl substituted monomers (FIG. 1).Phosphordiester linkages may connect the individual monomers, butmodified linkages like phosphorothioate linkages and other linkagesknown to a person skilled in the field [Nawrot and Sipa, Curr. TopicsMed. Chem. 2006, 6, 913-925] may be used instead. The RNA complexes maycomprise two strands that together constitute an siRNA duplex composedof an antisense strand (the antisense strand is also herein referred toas the guide strand) and a passenger strand (the passenger strand isalso herein referred to as the sense strand), but a single strandedmicroRNA mimicking molecule is also considered herein as an RNA complexof the invention, as is a single stranded antisense molecule that forexample is useful for targeting microRNAs.

In the embodiments of the invention, the RNA complex comprises one ormore hydroxymethyl modified nucleotide monomer(s) (see FIG. 1).Hereunder as one such example is an acyclic nucleotide monomer, morepreferably an acyclic monomer selected from the group consisting ofmonomers D-J. Thus, the embodiments described in the first aspect withregards to hydroxymethyl modified nucleotide monomers will apply forother embodiments relating to acyclic nucleotide monomers.

The use of hydroxymethyl modified nucleotide monomers may be favouredfor several reasons. They may e.g. be used to increase gene silencingeffect of the RNA complexes and the incorporation of one or morehydroxymethyl modified nucleotide monomer(s), for example towards theends of the RNA complexes induce significant stability towardsnucleolytic degradation. They may also be used to decrease the genesilencing effect of the passenger strand of an siRNA complex thusreducing the number of off-target effects.

In one preferred embodiment of the invention, the RNA complex comprisingone or more hydroxymethyl modified nucleotide monomer(s) is a singlestranded RNA construct.

In one preferred embodiment of the invention, the RNA complex comprisingone or more hydroxymethyl modified nucleotide monomer(s) is a singlestranded RNA construct that is able to inhibit gene expression by actingas a single stranded antisense molecule.

In one preferred embodiment of the invention, the RNA complex comprisingone or more hydroxymethyl modified nucleotide monomer(s) is a singlestranded RNA construct that functionally mimics a microRNA.

In one preferred embodiment of the invention, the RNA complex comprisingone or more hydroxymethyl modified nucleotide monomer(s) is an siRNAconstruct.

Accordingly, in one embodiment, the antisense strand of an siRNAconstruct comprises one or more hydroxymethyl modified nucleotidemonomer(s).

In another embodiment, the passenger strand of an siRNA constructcomprises one or more hydroxymethyl modified nucleotide monomer(s). Inyet another embodiment, a first and second RNA molecule of a nickedpassenger strand of an siRNA construct each contain one or morehydroxymethyl modified nucleotide monomer(s).

In one embodiment of the invention, the number of hydroxymethyl modifiednucleotide monomers in the antisense strand is 10. In other embodimentsof the invention, the number of hydroxymethyl modified nucleotidemonomer(s) in the antisense strand is 9, 8, 7, 6, 5, 4, 3, 2 or 1,respectively.

In another embodiment, all nucleotides of the antisense strand arehydroxymethyl modified nucleotide monomers.

In a preferred embodiment, all hydroxymethyl modified nucleotidemonomers in the antisense strand is present in positions 1-8, whereinthe positions are counted from the 5′end. Even more preferably, thehydroxymethyl modified nucleotide monomers in the antisense strand ispresent in positions 2-7 corresponding to the so-called seed region of amicroRNA. Thus, presence of hydroxymethyl modified nucleotide monomersin the aforementioned regions will prevent the antisense strand fromacting as a microRNA, which reduces off target effects when theantisense strand is intended to function as siRNA.

In a preferred embodiment, at least one hydroxymethyl modifiednucleotide monomer is present in one of positions 9-16, wherein thepositions are counted from the 5′end. Even more preferred is thepresence of 2, 3, 4, 5 or 6 hydroxymethyl modified nucleotide monomer ispresent in positions 9-16 and in another embodiemnt, hydroxymethylmodified nucleotide monomers in the antisense strand is present in allof positions 9-16,. In one embodiment, hydroxymethyl modified nucleotidemonomer are only present in regions 9-16 and not in the rest of theantisense strand.

Even more preferably, the hydroxymethyl modified nucleotide monomers inthe antisense strand is present in position 9-11 and preferably, not inthe rest of the oligonucleotide. The presence of hydroxymethyl modifiednucleotide monomers in the aforementioned regions will induce theantisense strand to act as a microRNA, i.e. ensure that the siRNA effectwill be minimal and the microRNA effect much higher. This effect likelystems from the reduced tendency towards full length binding because ofreduced affinity caused by the presence of an acyclic hydroxymethylsubstituted monomer, e.g. monomer D.

Likewise, in another embodiment of the invention, the number ofhydroxymethyl modified nucleotide monomers in the passenger strand of ansiRNA complex of the invention is 10. In other embodiments of theinvention, the number of hydroxymethyl modified nucleotide monomers inthe passenger strand of an siRNA complex of the invention is 9, 8, 7, 6,5, 4, 3, 2 or 1, respectively.

In another embodiment, all nucleotides of the passenger strand of ansiRNA complex of the invention are hydroxymethyl modified nucleotidemonomers.

In one embodiment, both the antisense strand and the passenger strand ofan siRNA complex of the invention contain one or more hydroxymethylmodified nucleotide monomer(s).

In one aspect, the present invention provides an RNA complex capable ofmediating nucleic acid modifications of a target nucleic acid. Such RNAcomplex may e.g. be a siRNA, microRNA or microRNA precursor(pre-microRNA).

The RNA complex of an siRNA complex of the invention comprises a coredouble stranded region comprising an antisense strand and a passengerstrand that is hybridised to the antisense strand.

A target nucleic acid as referred to in the present context is a nucleicacid, which has significant complementarity to the antisense strand ofthe complex. Preferably, complementarity is perfect over a stretch ofseveral nucleotides.

Thus, in one embodiment, complementarity is perfect over a stretch of 25nucleotides.

In other embodiments, complementarity is perfect over a stretch of 24nucleotides, 23 nucleotides, 22 nucleotides, 21 nucleotides, 20nucleotides, 19 nucleotides, 18 nucleotides, 17 nucleotides, 16nucleotides, 15 nucleotides, 14 nucleotides, 13 nucleotides, 12nucleotides, 11 nucleotides, 10 nucleotides, 9 nucleotides, 8nucleotides, 7 nucleotides or 6 nucleotides, respectively.

In one embodiment, the stretch of complementarity comprises 1 mismatch.In other embodiments, the stretch of complementarity comprises 2mismatches, 3 mismatches or 4 mismatches, respectively. A mismatch of 1is a region in the stretch of complementarity where a base pair cannotform, e.g. when G is opposite to A. When more mismatches are presentthey may be adjacent to each other or they may be spaced in differentregions of the stretch of complementarity.

The RNA complex of an siRNA complex of the invention comprises in apreferred embodiment a core double-stranded region, which is asubstantially double-stranded region. Single-stranded regions in the RNAcomplex are primarily related to overhangs of the complex.

Thus, in one embodiment, the double-stranded region of an siRNA complexof the invention comprises 1 mismatch. In other embodiments, thedouble-stranded region comprises 2 mismatches, 3 mismatches and 4mismatches, respectively.

As used herein, the term “target nucleic acid” encompasses any RNA/DNAthat would be subject to modulation guided by the antisense strand, suchas targeted cleavage or steric blockage. The target RNA/DNA could, forexample be genomic DNA, genomic viral RNA, mRNA, a pre-mRNA, or anon-coding RNA

As used herein, the term “target nucleic acid modification” means anymodification to a target nucleic acid, including those that affect theactivity of the target nucleic acid, without affecting the structure ofthe target nucleic acid.

A preferred target nucleic acid of the invention is mRNA. Accordingly,in one embodiment the nucleic acid modification mediated by the RNAcomplex is RNA interference (RNAi). In a preferred embodiment, RNAimediates degradation of the mRNA. In another preferred embodiment, RNAimediates translational inhibition of the mRNA. In another embodiment,the RNAi mediates both translational inhibition and degradation of themRNA.

In other preferred embodiments, the target nucleic acid is a non-codingRNA, e.g. a tRNA, miRNA, snRNA, snoRNA or an rRNA.

In still another embodiment, the target nucleic acid is genomic DNA. Insuch embodiments, preferred nucleic acid modifications include DNAmethylation and DNA deletion.

The size of the RNA complex of the invention can be varied while stillfulfilling one or more objects of the invention. This e.g. applies wherethe particular object is reduced off-target effect.

Thus, the core double-stranded region of an siRNA complex of theinvention may comprise a number of base pairs selected from the group of10 base pairs, 11 base pairs, 12 base pairs, 13 base pairs, 14 basepairs, 15 base pairs, 16 base pairs, 17 base pairs, 18 base pairs, 19base pairs, 20 base pairs, 21 base pairs, 22 base pairs, 23 base pairs,24 base pairs and 25 base pairs, 26 base pairs, 27 base pairs, 28 basepairs, 29 base pairs, 30 base pairs, 35 base pairs, 40 base pairs, 42base pairs, 45 base pairs, 50 base pairs, 55 base pairs, 60 base pairsor 62 base pairs.

In one embodiment, the core double stranded region of an siRNA complexof the invention comprises from 15 to 40 base pairs.

In another preferred embodiment, the core double stranded region of ansiRNA complex of the invention comprises 18-22 base pairs.

In one embodiment, the core double stranded region of an siRNA complexof the invention is even longer than 40 base pairs, although it is knownthat in some cells, the introduction of longer double stranded RNAcomplex may induce an interferon dependent non-specific response. In onesuch embodiment, it is contemplated that the complex is processed toshorter double-stranded RNA complexes before engaging with a RGPC. AnRNase III like enzyme such as DICER may execute processing. Dicer alsoprocesses double stranded RNA shorter than 40 base pairs and such RNAcomplexes (referred to as Dicer substrates) have various advantages ascompared to siRNA that enters RISC without processing. Hence, in oneembodiment, the RNA complexes of the invention are Dicer substrates.

In another embodiment, the RNA complex is single stranded and has nodouble stranded region.

In yet another embodiment, the RNA complex is single stranded but foldssuch that it contains one or more double stranded regions. Suchembodiments are useful e.g. for mimicking microRNAs and their functions.

In yet another embodiment, the core double stranded region of an siRNAcomplex of the invention is shorter than 10 base pairs and thuscomprises from one to nine base pairs.

In one embodiment of the invention, the core double stranded region ofthe RNA complex is comprised by more than two RNA strands.

In one embodiment of the invention, the core double stranded region ofthe RNA complex is comprised by three RNA strands.

In another embodiment of the invention, the core double stranded regionof the RNA complex is comprised by four or more RNA strands.

In a preferred embodiment of the invention, the siRNA complex of theinvention comprises overhangs. An overhang as used in the presentcontext refers to a short single-stranded region following adouble-stranded region.

In one embodiment, the antisense strand of an siRNA complex of theinvention comprises a 3′-overhang.

In another embodiment, the passenger strand of an siRNA complex of theinvention comprises a 3′-overhang.

In yet another embodiment, the antisense strand of an siRNA complex ofthe invention comprises a 5′-overhang.

In still another embodiment, the passenger strand of an siRNA complex ofthe invention comprises a 5′-overhang.

In a preferred embodiment, both the antisense strand and the passengerstrand of an siRNA complex of the invention comprise a 3′-overhang.

The overhangs of an siRNA complex of the invention can be of varyinglength, without interfering with the basic function of the complex.Thus, in one embodiment the overhangs are selected from the group ofoverhangs with a length of 1 nucleotide, 2 nucleotides, 3 nucleotides, 4nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides and 8nucleotides.

Most preferred overhangs of an siRNA complex of the invention areoverhangs with a length of 1, 2 and 3 nucleotides, respectively.

In one embodiment, the overhang of the antisense strand of an siRNAcomplex of the invention has the same length as the overhang of thepassenger strand.

In another embodiment, the overhang of the antisense strand of an siRNAcomplex of the invention does not have the same length as the overhangof the passenger strand

In still another embodiment of an siRNA complex of the invention, theRNA complex comprises at least one blunt end. A “blunt end” refers to anend of a double-stranded nucleic acid, which does not have anyprotruding nucleotides, i.e. both strands of the double-stranded nucleicacid ends at the same position.

In another embodiment, the siRNA complex of the invention is blunt endedat both ends.

Preferred RNA complexes of the invention are similar in overallstructure to the products of DICER processing of longer double strandedRNA complexes.

In another embodiment, the RNA complexes of the invention are Dicersubstrates as mentioned above.

Other preferred RNA complexes of the invention are complexes wherein thecore double-stranded region comprises 18-22 base pairs, and wherein theantisense strand and the passenger strand each comprise a 3′-overhang of1-3 nucleotides.

The antisense strand of the RNA complex of the invention can havevarying lengths, without interfering with the function of the complex.Thus, in preferred embodiments, the antisense strand is an 8-mer, 9-mer,10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer,9-mer, 20-mer, 21-mer, 22-mer, 23-mer, a 24-mer, a 25-mer, a 26-mer, a27-mer, a 28-mer, 29-mer, 30-mer, 31-mer, 32-mer, 33-mer, 34-mer,35-mer, 36-mer, 37-mer, 38-mer, 39-mer, 40-mer, 41-mer, 42-mer, 43-mer,44-mer, 45-mer, 46-mer, 47-mer, 48-mer, 49-mer, 50-mer, 51-mer, 52-mer,53-mer, 54-mer, 55-mer, 56-mer, 57-mer, 58-mer, 59-mer, 60-mer, 61-meror a 62-mer, respectively. It is to be understood that e.g. a 19-mer isan antisense strand of 19 monomers, i.e. 19 nucleotides.

In another preferred embodiment, the antisense strand of the RNA complexis selected from the following group of antisense strands: A 15-mer,16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer and a 23-mer. Inone embodiment the passenger strand of an siRNA complex of the inventionis discontinuous. In one embodiment of an siRNA complex of theinvention, the passenger strand comprises several separate RNAmolecules. The number of RNA molecules may be 1, 2, 3, 4, 5 or 6.

In one embodiment, the length of individual RNA molecules of thepassenger strand of an siRNA complex of the invention is above 4monomers. In other embodiments, the length of individual RNA moleculesof the passenger strand is above 5 monomers, 6 monomers, 7 monomers, 8monomers, 9 monomers, 10 monomers, 11 monomers and 12 monomers,respectively.

In other embodiments, the length of individual RNA molecules of thepassenger strand of an siRNA complex of the invention is below 5monomers, 6 monomers, 7 monomers, 8 monomers, 9 monomers, 10 monomers,11 monomers and 12 monomers, respectively.

In one embodiment of the invention, a discontinuous passenger strand ofan siRNA complex of the invention comprises a first and a secondRNA-molecule, which together forms the discontinuous passenger strand,wherein the first RNA molecule is hybridised to the downstream part ofthe antisense strand and the second RNA molecule is hybridised to theupstream part of the antisense strand.

In one embodiment, the antisense strand of an siRNA complex of theinvention is discontinuous. Preferred discontinuities of the antisensestrands are the same as the preferred discontinuities of the passengerstrand.

A discontinuity of one of the strands of an siRNA complex of theinvention can be a nick. A nick is to be understood as a discontinuityin one strand of a double-stranded nucleic acid caused by a missingphosphodiester bond, however, without the double-stranded nucleic acidmissing a nucleotide. Thus, the bases opposite to the nick will still behybridised to bases on the nicked strand.

Another discontinuity of one of the strands of an siRNA complex of theinvention is an alternative nick, which is understood as a discontinuityin one strand of a double-stranded nucleic acid caused by one missingbond, or more than one missing bond in the sugar-phosphate backbone,other than a phosphodiester bond, however, without the double-strandednucleic acid missing a nucleobase. Thus, the bases opposite to the nickmay still be hybridised to bases on the nicked strand.

A gap as used as a nomination when an RNA strand of an RNA complex ofthe invention can be described to have a discontinuity where at leastone nucleotide or nucleoside or a nucleobase is missing in thedouble-stranded nucleic acid.

Preferably, the 5′-ends of the RNA complex is phosphorylated or isavailable for phosphorylation. Available for phosphorylation means thatthe 5′-hydroxy group has not been blocked e.g. by direct conjugation orby other conjugation to other groups in the vicinity of the 5′-hydroxygroup, which will prevent the 5′-hydroxy group from beingphosphorylated.

Hence, in a preferred embodiment of the invention, the RNA molecule(s)of the RNA complex comprise(s) a 5′-end phosphate and a 3′-hydroxygroup.

In another embodiment, the second RNA molecule of an siRNA complex ofthe invention comprises a 5′-end phosphate and a 3′-hydroxy group.

In yet another embodiment, the antisense strand comprises a 5′-endphosphate and a 3′-hydroxy group.

In some embodiments of the invention, it is preferred that the RNAcomplex comprises nucleotide analogues other than the hydroxymethylmodified nucleotides. Such nucleotide analogues other than thehydroxymethyl modified nucleotides are termed below as “alternativelymodified nucleotides”.

The use of alternatively modified nucleotides may be favoured forseveral reasons. They may e.g. be used to increase the meltingtemperature of the core double stranded region of an siRNA complex ofthe invention.

The use of alternatively modified nucleotides may be favoured toincrease the melting temperature of the double stranded structure formedbetween the antisense strand and the target nucleic acid.

Accordingly, in one embodiment, the antisense strand comprisesalternatively modified nucleotides.

In another embodiment, the passenger strand of an siRNA complex of theinvention comprises alternatively modified nucleotides.

In yet another embodiment, a first and second RNA molecule of thepassenger strand of an siRNA complex of the invention each containalternatively modified nucleotides.

In one embodiment of the invention, the number of alternatively modifiednucleotides in the RNA complex is 10. In other embodiments of theinvention, the number of nucleotide analogues in the RNA complex is 9,8, 7, 6, 5, 4, 3, 2 or 1, respectively.

In one embodiment of the invention, the number of alternatively modifiednucleotides in the antisense strand is 10. In other embodiments of theinvention, the number of nucleotide analogues in the antisense strand is9, 8, 7, 6, 5, 4, 3, 2 or 1, respectively.

In another embodiment, all nucleotides of the antisense strand arealternatively modified nucleotides or a combination of alternativelymodified nucleotides and hydroxymethyl-substituted nucleotides.

Likewise, in another embodiment of the invention, the number ofnucleotide analogues in the passenger strand of an siRNA complex of theinvention is 10. In other embodiments of the invention, the number ofnucleotide analogues in the passenger strand is 9, 8, 7, 6, 5, 4, 3, 2or 1, respectively.

In another embodiment, all nucleotides of the passenger strand of ansiRNA complex of the invention are nucleotide analogues or a combinationof alternatively modified nucleotides and hydroxymethyl-substitutednucleotides.

In one embodiment, both the antisense strand and the passenger strand ofan siRNA complex of the invention contain alternatively modifiednucleotides.

In one embodiment, the alternatively modified nucleotides of the RNAcomplex are identical, i.e. they are for example all LNA or all2′-O-Me-RNA. In another embodiment, various different alternativelymodified nucleotides are used in the same RNA complex.

In one embodiment, the RNA complex comprises phosphorothioate linkages.

In another embodiment, the RNA complex comprises a mixture of naturalphosphordiester and phosphorothioate linkages.

Preferred nucleotide analogues of the invention is nucleotide analoguesselected from the group of 2′-O-alkyl-RNA monomers, 2′-amino-DNAmonomers, 2′-fluoro-DNA monomers, LNA monomers, HNA monomers, ANAmonomers, FANA monomer, DNA monomers, PNA monomers and INA monomers, butother monomers can also be used [Nawrot and Sipa, Curr. Topics Med.Chem. 2006, 6, 913-925].

In one embodiment the hydroxymethyl substituent of the hydroxymethylsubstituted monomers of the invention is functionalised by a conjugatinggroup. A conjugating group is a group known to a Person skilled in theart that changes, expands or improves the properties of an RNA complexof the invention. Such groups may be useful for modulating cellulardistribution, organ distribution, tissue distribution, duplex meltingtemperatures, target affinity, biostability, signalling of hybridizationetc.

In one embodiment the hydroxymethyl substituent of the hydroxymethylsubstituted monomers of the invention is functionalised by an etherlinkage between a conjugated group and the methylene group of thehydroxymethyl substituent. See FIG. 2 (Monomer F).

In one embodiment the hydroxymethyl substituent of the hydroxymethylsubstituted monomers of the invention is converted into a thioetherfunctionality before incorporation into the RNA complex of the inventionusing methods known to a Person skilled in the art. See FIG. 2 (MonomerG).

In another embodiment the hydroxymethyl substituent of the hydroxymethylsubstituted monomers of the invention is converted into a mercaptomethylfunctionality before incorporation into the RNA complex of the inventionusing methods known to a Person skilled in the art. See FIG. 2 (MonomerG, R=H). This mercapto functionality is properly protected as e.g. itsacetyl derivative during RNA synthesis using methods know to a Personskilled in the art.

In one embodiment the hydroxymethyl substituent of the hydroxymethylsubstituted monomers of the invention is converted into an aminefunctionality before incorporation into the RNA complex of the inventionusing methods known to a Person skilled in the art. See FIG. 2 (MonomerG, R=H). This amine functionality is properly protected as e.g. itstrifluoroacetyl or Fmoc derivative during RNA synthesis using methodsknow to a Person skilled in the art.

In one embodiment the hydroxymethyl substituent of the hydroxymethylsubstituted monomers of the invention is acting as a handle forattachment of amide-linked conjugating groups. This involves conversionof the hydroxyl unit of the hydroxymethyl substituent into an amineunit, for example as described above, and further derivatisation of thisamino group by e.g. a conjugating group by amide bond formation usingmethods known to a Person skilled in the art. This may take place beforeRNA synthesis or after RNA synthesis using methods known to a Personskilled in the art (FIG. 2, Monomer H)

In one embodiment the hydroxymethyl substituent of the hydroxymethylsubstituted monomers of the invention is acting as a handle forattachment of amino-linked conjugating groups. This involves conversionof the hydroxyl unit of the hydroxymethyl substituent into an amineunit, for example as described above, and further derivatisation of thisamino group by e.g. a conjugating group by amine bond formation usingmethods known to a Person skilled in the art. This may take place beforeRNA synthesis or after RNA synthesis using methods known to a Personskilled in the art (FIG. 2, Monomer I).

In still one embodiment, the amine group used for conjugation is anamino group, a piperazino group or a diamino alkyl group. Such monomersare called amine-derivatised monomers. Each of these groups may befurther derivatised or conjugated (FIG. 2, Monomer J).

In one embodiment, the RNA complex of the invention has reduced offtarget effects as compared to native RNA complexes.

In one preferred embodiment, the RNA complex has at least onehydroxymethyl-substituted monomer of the invention in the antisensestrand.

In another preferred embodiment, the RNA complex has at least onehydroxymethyl-substituted monomer of the invention incorporated in oraround the so-called seed region of the antisense strand, i.e. in atleast one of positions no. 1-12 from the 5′-end of the antisense strand.

In yet another preferred embodiment, the RNA complex has at least onehydroxymethyl-substituted monomer of the invention incorporated in atleast one of positions no. 2-10 from the 5′-end of the antisense strand.

In yet another preferred embodiment, the RNA complex has onehydroxymethyl-substituted monomer of the invention incorporated in oneof positions no. 3-8 from the 5′-end of the antisense strand.

In yet another preferred embodiment, the RNA complex has onehydroxymethyl-substituted monomer of the invention incorporated in oneof positions no. 7 or 8 from the 5′-end of the antisense strand.

In yet another preferred embodiment, the RNA complex has onehydroxymethyl-substituted monomer of the invention incorporated inposition no. 7 from the 5′-end of the antisense strand.

In yet another preferred embodiment, the RNA complex has onehydroxymethyl-substituted monomer of the invention incorporated inpositions no. 9-16 from the 5′-end of the antisense strand.

In yet another preferred embodiment, the RNA complex has onehydroxymethyl-substituted monomer of the invention incorporated inpositions no. 9-11 from the 5′-end of the antisense strand.

In yet another preferred embodiment, the RNA complex has onehydroxymethyl-substituted monomer of the invention incorporated inpositions no. 9-10 from the 5′-end of the antisense strand.

In another embodiment, the RNA complex of the invention produces areduced immune response as compared to native RNA complexes.

In still another embodiment, the RNA complexes of the invention have aprolonged effect as compared to native RNA complexes.

In yet another embodiment, the RNA complexes of the invention have anincreased effect as compared to native RNA complexes. Accordingly, in apreferred embodiment, the RNA complex mediate RNAi more effectively thanthe native RNA complex, e.g. by more efficient degradation of targetmRNA or by more efficient translational inhibition of target mRNA.

In still another embodiment, the RNA complexes of the invention aredelivered efficiently to specific organs or tissues of a human or ananimal.

In yet still another embodiment, the RNA complexes of the invention areable to penetrate the cell membrane efficiently. In yet still anotherembodiment, the RNA complexes of the invention are able to penetrate thecell membrane more efficiently that natural RNA complexes.

In one embodiment, the RNA complexes of the invention are able to bindto plasma proteins which increases the retention of the RNA complexes inthe human body.

Second Aspect, Preparation of RNA Complex

Another aspect of the invention is a method of preparing a two strandedRNA complex of the invention comprising incubating the antisense strandwith the passenger strand under conditions wherein a RNA complexcomprising a core double stranded region is formed, said RNA complexbeing capable of mediating RNA interference of a corresponding cellularRNA.

In alternative embodiments of this aspect, the RNA complex issubstituted by an RNA duplex of the invention (tenth aspect).

Third Aspect, Method of Mediating Nucleic Acid Modification

Still another aspect of the invention is a method of mediating nucleicacid modification of a target nucleic acid in a cell or an organismcomprising the steps:

-   -   a. Contacting a cell or organism with the RNA complex of the        invention under conditions wherein modification of a target        nucleic acid can occur    -   b. Thereby mediating modification of a target nucleic acid

In preferred embodiments, the method of mediating nucleic acidmodification of a target nucleic acid is performed in vitro.

In preferred embodiments, the method of mediating nucleic acidmodification of a target nucleic acid is performed in vivo, i.e. inanimals, in humans or in non-human animals.

In preferred embodiments, the method of mediating nucleic acidmodification of a target nucleic acid is performed in cell cultures.

In yet another embodiment, the method is performed on an isolated cell.

In a preferred embodiment, the nucleic acid modification of the methodis RNA interference, preferable degradation of target mRNA ortranslational inhibition of target mRNA or inhibition of other types ofRNA, e.g. non-coding RNA.

In another embodiment, the nucleic acid modification is DNA methylation.

In alternative embodiments of this aspect, the RNA complex issubstituted by either an oligonucleotide of the invention (ninth aspect)or an RNA duplex of the invention (tenth aspect).

Fourth Aspect, Method of Examining Gene Function

Another aspect of the invention is a method of examining the function ofa gene in a cell or organism comprising:

-   -   a. Introducing a RNA complex of the invention corresponding to        said gene into the cell or organism, thereby producing a test        cell or test organism    -   b. Maintaining the test cell or test organism under conditions        under which modification of a target nucleic acid can occur    -   c. Observing the phenotype of the test cell or organism produced        in step b and optionally comparing the observed phenotype with        the phenotype of an appropriate control cell or control        organism, thereby providing information about the function of        the gene.

The RNA complex of the invention can be introduced into cells e.g. usingtransfection, as outlined in the appended examples.

The phenotypeof the organism or cell may be observed e.g. usingproteomics to assess protein levels or using microarrays to assess RNAlevels. Also a more defined phenotype may be used, e.g. the expressionof one particular gene.

The information obtained about the function of a gene may be used todetermine whether a gene product is a suitable target for therapeuticintervention in relation to a particular disease. Thus, if it isdemonstrated that a certain gene product act in a certain biochemicalpathway known to be affected in e.g. a specific subtype of cancer, thegene product might be a suitable target for therapeutic intervention fortreatment of the aforementioned subtype of cancer.

In a preferred embodiment of the method of examining the function of agene in a cell or organism, the nucleic acid modifications of the methodare RNA interference, preferable degradation of target mRNA ortranslational inhibition of target RNA.

In another embodiment, the nucleic acid modification is DNA methylation.

In preferred embodiments of the method of examining the function of agene in a cell or organism, the method is performed in cell cultures, invitro or in vivo.

In yet another embodiment, the method is performed on an isolated cell.

In alternative embodiments of this aspect, the RNA complex issubstituted by either an oligonucleotide of the invention (ninth aspect)or an RNA duplex of the invention (tenth aspect).

Fifth Aspect, Method of Evaluating Agent

Another aspect of the invention is a method of assessing whether anagent acts on a gene product comprising the steps:

-   -   a. Introducing the RNA complex of the invention corresponding to        said gene into a cell or organism, thereby producing a test cell        or test organism    -   b. Maintaining the test cell or test organism under conditions        under which modification of a target nucleic acid occurs    -   c. Introducing the agent into the test cell or test organism    -   d. Observing the phenotype of the test cell or organism produced        in step c and optionally comparing the observed phenotype with        the phenotype of an appropriate control cell or control        organism, thereby providing information about whether the agent        acts on the gene product

A preferred control in step d is a test cell or test organism that hasnot had the RNA complex of step a introduced.

In a preferred embodiment of the method of assessing whether an agentacts on a gene or gene product, the nucleic acid modifications of themethod are RNA interference, preferable degradation of target RNA ortranslational inhibition of target RNA. In another embodiment,modification of nucleic acid modifications is DNA methylation.

In preferred embodiments of the method of assessing whether an agentacts on a gene product, the method is performed in cell cultures, invitro or in vivo.

In yet another embodiment, the method is performed on an isolated cell.

In alternative embodiments of this aspect, the RNA complex issubstituted by either an oligonucleotide of the invention (ninth aspect)or an RNA duplex of the invention (tenth aspect).

Sixth Aspect, Pharmaceutical Composition

Still another aspect of the invention is the RNA complex and apharmaceutically acceptable diluent, carrier or adjuvant. It will beapparent to the skilled man that the RNA complexes of the invention canbe designed to target specific genes and gene products. It is to beunderstood that the RNA complexes will target a DNA sequence or a RNAsequence, and not a protein. However, the level of a gene product suchas a protein may be affected indirectly, if its mRNA or a non-coding RNAis modified e.g. by RNA degradation or translational inhibition. Alsothe expression of the gene encoding the protein may be affected, e.g.because of DNA methylation.

In alternative embodiments of this aspect, the RNA complex issubstituted by either an oligonucleotide of the invention (ninth aspect)or an RNA duplex of the invention (tenth aspect).

Seventh Aspect, use a Medicament

Thus, another aspect is the RNA complex of the invention for use as amedicament. Once a therapeutic target has been validated, the skilledman can design RNA complexes that affect the level and the activity ofthe target, because the specificity of the RNA complexes liesexclusively within the sequence of the antisense strand. For native RNAcomplexes with a continuous passenger strand, there remains a problemwith off-target effects due to the passenger strand acting as a guidesequence.

In alternative embodiments of this aspect, the RNA complex issubstituted by either an oligonucleotide of the invention (ninth aspect)or an RNA duplex of the invention (tenth aspect).

Eighth Aspect, Monomers

An aspect of the invention is monomers suitable for incorporation of thehydroxymethyl substituted monomers of the invention and methods fortheir preparation from readily available starting materials. Thymin-1-ylderivatives of hydroxymethyl substituted monomers of the invention havebeen incorporated into DNA strands, and procedures for preparation oftheir phosphoramidite building blocks for automated DNA/RNA synthesishave been reported [K. D. Nielsen et al., Bioorg. Med. Chem. 1995, 3,1493; H. Thrane et al., Tetrahedron 1995, 51, 10389; P. Nielsen et al.,Bioorg. Med. Chem. 1995, 3, 19].

Most often, the RNA complexes of the invention will be prepared byautomated oligonucleotide synthesis as known to a Person skilled in theart.

The incorporation of the hydroxymethyl substituted monomers of theinvention into the RNA complexes of the invention follows standardmethods for a) RNA synthesis on an automated RNA synthesizer, b) RNAwork-up, c) RNA purification and d) RNA isolation [F. Eckstein,Oligonucleotides and Analogues, IRL Press, Oxford University Press,1991]. The hydroxymethyl substituted RNA oligonucleotides (=RNA strands)and RNA complexes can be synthesised using phosphoramidite derivativesusing the standard techniques for RNA synthesis.

In a preferred embodiment, methods of preparation of the phosphoramiditederivatives of the hydroxymethyl substituted monomers of the inventionbegins from a ribonucleoside, for example a O5′-DMT protected derivativeof a ribonucleoside that for the bases adenine, guanine, cytosine and5-methylcytosine contains base protecting groups like for example,benzoyl, isobutyryl, acetyl, phenoxyacetyl, tert-butylphenoxyacetyl orother standard base protecting groups known to a Person skilled in theart.

In a preferred embodiment, the invention comprises methods to preparemonomeric building blocks suitable for incorporation of the Monomers Dand E having a 2′,3′-cleaved carbon-carbon bond (ribonucleosidenomenclature).

In other preferred embodiments, the invention comprises methods toprepare monomeric building blocks suitable for incorporation of theMonomers like F-J having a 2′,3′-cleaved carbon-carbon bond and inaddition carrying a functionality or group at for example its 2′-carbonatom (ribonucleoside nomenclature) other than a hydroxy group.

In a preferred embodiment of the invention, the method of preparation ofthe phosphoramidite derivatives of Monomer D comprises among the keysteps 2′,3′-glycol cleavage, reduction of the resulting intermediate,selective O2′-protection and O3′-phosphitylation.

In a preferred embodiment the 2′,3′-glycol cleavage is undertaken usingoxidative cleavage with for example sodium Periodate as reagent.

In another preferred embodiment the reduction of the intermediate aftersodium Periodate cleavage is reduced to the corresponding diol effectedby for example sodium borohydride.

For incorporation of Monomer D into the RNA complexes of the inventionit is necessary to protect the 2′-hydroxy group (ribonucleosidenomenclature). In a preferred embodiment of the invention this is doneby benzoylation. It may be beneficial to use only slightly more than oneequivalent of benzoylation reagent (benzoyl chloride or e.g. benzoylanhydride) in order to optimise the selectivity of the protection, i.e.the amount of O2′-benzoylation relative to O3′-benzoylation. In onepreferred embodiment the benzoylation is performed below roomtemperature. In another useful embodiment the benzoylation is performedbelow 0° C. or even below -50° C.

In another preferred embodiment the O2′-protection is done byacetylation or by Performing acylation using an acylation reagent knownto a Person skilled in the art of organic synthesis.

In another preferred embodiment the O2′-protection is done by silylationusing a silylation reagent and method known to a Person skilled in theart of organic synthesis. A preferred silylation protecting group istert-butyldimethylsilyl.

The subsequent phosphitylation reaction is in a preferred embodimentperformed using either the so-called “PCI” reagent[PCl(OCH₂CH₂CN)(N(iPr)₂)] or the so-called “bis-amidite” reagent[P(OCH₂CH₂CN)(N(iPr)₂)₂].

In a preferred embodiment of the methods of preparation of thephosphoramidite derivatives of Monomer D, the starting material is aribonucleoside, for example a O5′-DMT protected derivative of aribonucleoside that for the bases adenine, guanine, cytosine and5-methylcytosine contains base protecting groups like for example,benzoyl, isobutyryl, acetyl, phenoxyacetyl, tert-butylphenoxyacetyl orother standard base protecting groups known to a Person skilled in theart.

In another preferred embodiment, the invention provides a method ofpreparation of a phosphoramidite derivative of Monomer E.

In a preferred embodiment of the invention, the method of preparation ofthe phosphoramidite derivatives of Monomer E comprises among the keysteps 2′,3′-glycol cleavage, reduction of the resulting intermediate,selective O3′-protection and O2′-phosphitylation. The O3′-protection canfor example be performed by silylation or acylation, or by a combinationlike first O2′-benzoylation, then O3′-silylation, and thenO2′-debenzoylation. Other protecting groups may also be applied as wouldbe clear for a Person skilled in the art.

In another preferred embodiments, the method to prepare monomericbuilding blocks suitable for incorporation of the Monomers like F-J,having a 2′,3′-cleaved carbon-carbon bond and in addition carrying afunctionality at its 2′-carbon atom (ribonucleoside nomenclature) otherthan a hydroxy group, comprises among the key steps starting from aribonucleoside (for example a O5′-DMT protected ribonucleosde)2′,3′-glycol cleavage, reduction of the resulting intermediate,selective O3′-protection, conversion of the 2′-hydroxy group,O3′-deprotection and O3′-phosphitylation. The O3′-protection can forexample be performed by silylation or acylation, or a combination of theboth like first O2′-benzoylation, then O3′-silylation, and thenO2′-debenzoylation. Other protecting groups may also be applied as wouldbe clear for a person skilled in the art. The conversion of the2′-hydroxy group into another group like amino, acylated amino,alkylated amino, dialkylated amino, carbamoylated amino, piperazino,acylated piperazino, alkylated piperazino, carbamoylated piperazino,mercapto, acylated mercapto, alkylated mercapto, disulfide, acylatedhydroxy, alkylated hydroxy, carbamoylated hydroxy, etc., or bysubstituted and/or protected derivatives of these groups, can beperformed using methods and procedures known to a person skilled in theart of organic synthesis. Such methods and procedures includesubstitution reactions on an activated derivative of the 2′-hydroxygroup or acylation or carbamoylation reactions. Such methods andprocedures also include O2′-alkylation reactions and alkylationreactions after inclusion of other C2′ attached groups like amino ormercapto. Yet another possibility is oxidation of the 2′-hydroxy groupto give an aldehyde functionality, which may be further modified by e.g.reaction with nucleophiles, or to give a carboxy functionality, whichmay be further modified by e.g. reaction with nucleophiles afterconversion of the carboxy functionality into an antivated derivativelike an active ester.

In another embodiment of the invention, the method to prepare monomericbuilding blocks suitable for incorporation of the Monomers like F-J, but“inversed” (like Monomers D and E can be considered “inversed”) suchthat the O2′ atom is phosphitylated and it is the 3′-hydroxy group thatis converted into another group such that the C3′ atom is linked to afunctionality other that a hydroxy group, comprises among the key stepsstarting from a ribonucleoside (for example a O5′-DMT protectedribonucleosde) 2′,3′-glycol cleavage, reduction of the resultingintermediate, selective O2′-protection, conversion of the 3′-hydroxygroup, O2′-deprotection and O2′-phosphitylation. The O2′-protection canfor example be performed by silylation or acylation, or a combination ofthe both. Other protecting groups may also be applied as would be clearfor a person skilled in the art. The conversion of the 3′-hydroxy groupinto another group like amino, acylated amino, alkylated amino,dialkylated amino, carbamoylated amino, piperazino, acylated piperazino,alkylated piperazino, carbamoylated piperazino, mercapto, acylatedmercapto, alkylated mercapto, disulfide, acylated hydroxy, alkylatedhydroxy, carbamoylated hydroxy, etc., or by substituted and/or protectedderivatives of these groups, can be performed using methods andprocedures known to a person skilled in the art of organic synthesis.Such methods and procedures include substitution reactions on anactivated derivative of the 3′-hydroxy group or acylation orcarbamoylation reactions. Such methods and procedures also includeO3′-alkylation reactions and alkylation reactions after inclusion ofother C3′ attached groups like amino or mercapto. Yet anotherpossibility is oxidation of the 3′-hydroxy group to give an aldehydefunctionality, which may be further modified by e.g. reaction withnucleophiles, or to give a carboxy functionality, which may be furthermodified by e.g. reaction with nucleophiles after conversion of thecarboxy functionality into an antivated derivative like an active ester.

In one embodiment, a 2′-C-piperazino derivative is prepared byconverting the 2′-hydroxy group into a leaving group (e.g. mesylatederivative) followed by reaction with a large excess of piperazine. Thisfor example can be performed as a step toward synthesis of aphosphoramidite of structure Amidite J (see figure below).

In yet another embodiment, the invention comprises methods to preparemonomeric building blocks suitable for incorporation of thehydroxymethyl substituted monomers of the invention carrying groups orfunctionalities at the C1′ atom (ribonucleoside nomenclature) that isdifferent from a natural nucleobase. Such groups or functionalities,that may contain protecting groups, include e.g. pyrene, perylene,fluorophores, hydrogen, alkyl, reactive groups and heterocycles otherthan the natural nucleobases.

In yet another embodiment, the invention comprises methods to preparemonomeric building blocks suitable for incorporation of thehydroxymethyl substituted monomers of the invention that are constitutedas H-phosphonate derivatives instead of phosphoramidite derivatives.

Below are shown examples of structures of some preferred embodiments ofthe invention with respect to phosphoramidite (=amidite) building blocks(DMT=4,4′-dimethoxytrityl; Base=natural nucleobase; CEtO=cyanoethoxy):

Ninth Aspect, Oligonucleotide Comprising Acyclic Oligonucleotides

A ninth aspect of the invention is an oligonucleotide comprising anacyclic nucleotide monomer. As will be apparent from the description andthe examples section such oligonucleotide has various uses andadvantages.

In a preferred embodiment, the acyclic nucleotide monomer is a2′-3′-seco-nucleotide monomer. Oligonucleotides of the inventioncomprising acyclic nucleotide monomers have surprisingly been found tobe substrates cellular enzymes of the RNAi machinery and in someinstances, these oligonucleotides are even better substrates than anidentical oligonucleotide without acyclic nucleotide monomers.

Preferably, the acyclic nucleotide monomer is selected from the groupconsisting of monomer E, F, G, H, I or J (see FIG. 1). As will be clearto the skilled man, G, F, H, I and J can all be made from syntheticprecursors of monomer D. As indicated in FIG. 2, the acyclic monomersmay transformed into derivatives carrying conjugating groups suchcholestoryl derivatives, alkyl, fluorophores, polyamines, amino acids,saccharides, polypeptides etc. Such conjugating groups may e.g. beuseful for better biostability and/or biodistribution when theoligonucleotide is used for modulating the activity of target mRNAs incells, organs or organisms.

The length of the oligonucleotide is preferably from 10 to 40 nucleotidemonomers. Even more preferred is a length from 18 to 30 nucleotidemonomers.

In a preferred embodiment, the oligonucleotide of the inventioncomprises less than 5 acyclic nucleotide monomers. In another preferredembodiment, the oligonucleotide comprises no more than 1 acyclicnucleotide monomer per 5 nucleotide monomers other than acyclicnucleotide monomers. Even more preferred is no more than 1 acyclicmonomer per 8 nucleotide monomers other than acyclic nucleotidemonomers. If the number of acyclic nucleotide monomer gets to high, thebinding affinity of the oligonucleotide of the invention to acomplementary nucleic acid is compromised.

Thus, in another embodiment, the oligonucleotide comprises from 1 to 5acyclic nucleotide monomers.

In a preferred embodiment, acyclic nucleotide monomers are only presentin one or more of position 1-8 and more preferably in positions 2-7 ofthe oligonucleotide. The positions are counted from the 5′end of theoligonucleotide. Acyclic nucleotide monomers in these regions willreduce or prevent the oligonucleotide from acting as a microRNA, asthese positions correspond to the so-called seed region of a microRNA.This is relevant e.g. where the oligonucleotide is intended to functionas the guide strand of an siRNA.

In a preferred embodiment, all hydroxymethyl modified nucleotidemonomers in the antisense strand is present in positions 9-16, whereinthe positions are counted from the 5′end. Even more preferably, thehydroxymethyl modified nucleotide monomers in the antisense strand ispresent in position 9-11. Thus, presence of hydroxymethyl modifiednucleotide monomers in the aforementioned regions will induce theantisense strand to act as a microRNA, i.e. ensure that the siRNA effectwill be minimal and the microRNA effect much higher. This effect likelystems from the reduced tendency towards full length binding because ofreduced affinity caused by the presence of an acyclic hydroxymethylsubstituted monomer, e.g. monomer D.

In a preferred embodiment, the oligonucleotide does not comprise DNAsequences of more than 8 consecutive DNA monomers. Even more preferredis no more than 6 consecutive DNA monomers and most preferably in nomore than 4 consecutive DNA monomers. Consecutive DNA monomers typicallywill enable the oligonucleotide to activate RNase H when bound to acomplementary RNA, which leads to degradation of the RNA. In someembodiments of the invention, this is not desirable. Thus, in a furtherembodiment, the oligonucleotide does not contain any DNA monomers atall.

In other embodiments, RNase H activation is desirable and it ispreferred that the oligonucleotide comprises more than 4 consecutive DNAmonomers, more preferably more 6 DNA monomers and most preferably morethan 8 DNA monomers.

In yet another embodiment, the oligonucleotide comprises more than 50%RNA monomers. A high degree of RNA monomers will facilitate interactionwith RNA-interacting proteins, e.g. by functioning as a substrate orguide (or co-factor) for a cellular enzyme such as RISC.

Thus, in another embodiment, it is preferred that more than 80% of themonomers of the oligonucleotide are RNA monomers. In yet anotherembodiment, it is preferred that more than 90% of the monomers of theoligonucleotide are RNA monomers.

As will be apparent, the oligonucleotide may also comprise nucleotidemonomer analogues. In one such embodiment, acyclic nucleotide monomersand RNA monomers make up more than 80% of all nucleotide monomers. Inanother embodiment, acyclic monomers and RNA monomers make up more than90% of all nucleotide monomers.

When the oligonucleotide comprises nucleotide monomer analogues, it ispreferred that they are selected from the group consisting of2′-O-alkyl-RNA monomers, 2′-amino-DNA monomers, 2′-fluoro-DNA monomers,LNA monomers, PNA monomers, HNA monomers, ANA monomers, FANA monomers,CeNA monomers, ENA monomers, DNA monomers and INA monomers. Nucleotideanalogues are typically used to modulate binding affinity, increasebiostability and in general give the oligonucleotide more drug-likeproperties.

In one embodiment, the oligonucleotide comprises at least 2 LNAnucleotide analogues. Acyclic nucleotide monomers typically decrease themelting temperature (i.e. binding affinity) of the oligonucleotide ofthe invention base paired to a complementary nucleic acid and LNAnucleotide monomers may be used to counteract this decrease in meltingtemperature. I.e. in one embodiment, the number of acyclic nucleotidemonomers is identical to the number of LNA nucleotide monomers.

In a preferred embodiment, the oligonucleotide comprises only acyclicmonomers and RNA monomers.

In another preferred embodiment, the oligonucleotide comprises onlyacyclic nucleotide monomers, RNA monomers, and LNA nucleotide analogues.

In a preferred embodiment, the oligonucleotide of the inventioncomprises one or more linkage(s) selected from the group consisting ofphosophorothioate linkage, boranophosphate linkage, ethylphosphonatelinkage, phosphoramidate linkage and phosphortriester linkage. Mostpreferred are a phosphorothioate linkage and/or a boranophosphatelinkage. These linkages improve the biostability of the oligonucleotideand have also been shown to have a positive effect on thebiodistribution of the oligonucleotide. In a preferred embodiment, theoligonucleotide comprises more than 50% of the aforementionedinternucleotide linkages and even more preferably more than 75%. In oneembodiment, all internucleotide linkages are of the aforementionedtypes.

In a preferred embodiment, the oligonucleotide of the invention is notbase paired to a complementary oligonucleotide, i.e. the oligonucleotideof the invention is single stranded.

In yet another embodiment, the oligonucleotide is capable of mediatingRISC dependent translational repression or degradation of target mRNAscomplementary to the oligonucleotide. The skilled man will recognizeRISC as the RNA Induced Silencing Complex and understand that in thisembodiment, the oligonucleotide will act as a guide sequence for RISCand thereby guide RISC to RNA oligonucleotides, typically mRNAs thatharbor partial or full complementarity to the oligonucleotide of theinvention. When the oligonucleotide guides RISC to mRNA targets ofpartial complementarity, the oligonucleotide may be seen as a microRNAmimic and when the oligonucleotide guides RISC to mRNA targets of fullcomplementarity; it may be seen as a single or double stranded siRNA.

RISC dependence may be assessed in cell lines by knocking out componentsof RISC using siRNA against the mRNAs encoding the RISC components andevaluate the activity of the oligonucleotide in the knock-out cell line.Such experiments are well known to those skilled in the art.

Tenth aspect, RNA duplex comprising oligonucleotide of invention

A tenth aspect of the invention is an RNA duplex comprising a firstoligonucleotide according to the invention and a second oligonucleotide.

In a preferred embodiment, the second oligonucleotide of the RNA duplexis also an oligonucleotide of the invention.

As will be clear, many of the features described with relation to theRNA complexes of the invention in the first aspect, are also applicableto RNA duplexes of the tenth aspect.

Preferably, the RNA duplex of the invention comprises a number of basepairs from 15 to 40 and in a preferred embodiment, comprises a number ofbase pairs selected from the group of 18 base pairs, 19 base pairs, 20base pairs, 21 base pairs, 22 base pairs and 23 base pairs.

In yet another embodiment, the RNA duplex comprises a number of basepairs from 25 to 30, more preferably from 26 to 28 and most preferably27 base pairs. Such RNA duplexes may be referred to as dicer substrateRNAs.

In a preferred embodiment, the RNA duplex of the invention comprises anoverhang.

In another embodiment, the RNA duplex comprises two overhangs.

In still another embodiment, the first oligonucleotide comprises a3′-overhang.

In still another embodiment, the second oligonucleotide comprises a3′-overhang.

Preferably, the length of the overhang is from 1 to 8 nucleotides andeven more preferably, the length of the overhang is selected from thegroup consisting of overhangs with a length of 1 nucleotide, 2nucleotides and 3 nucleotides.

In another embodiment, the RNA duplex comprises at least one blunt end.

In another embodiment, the RNA duplex is blunt ended in both ends.

In a preferred embodiment, the RNA duplex comprises a double-strandedregion of 18-22 base pairs, wherein the first oligonucleotide and thesecond oligonucleotide each comprise a 3′-overhang of 1-3 nucleotides.Such RNA duplex will be recognized as a canonical siRNA (shortinterfering RNA).

In one embodiment, one strand of the RNA duplex is discontinuous asdescribed in detail in the first aspect.

In one embodiment, the RNA duplex is capable of mediating translationalrepression or degradation of target mRNA complementary to the first orthe second oligonucleotide of the RNA duplex. I.e the RNA duplex willfunction as e.g. an siRNA, microRNA or pre-microRNA.

In one embodiment, the RNA duplex is capable of mediating translationalrepression or degradation of target mRNA while inducing reducedoff-target effects as compared to an identical RNA duplex with RNAmonomers instead of acyclic monomers. Reduced off targets may beachieved because of decreased binding affinity and also because eitherthe first or the second oligonucleotide may be modified such as to notbeing able to function as a guide strand for RISC. I.e. it can becontrolled which oligonucleotide of the RNA duplex function as passengerstrand (sense strand) and which will function as guide strand (antisensestrand).

In another embodiment, the RNA duplex is capable of mediatingtranslational repression or degradation of target mRNA while inducingreduced off-target effects when specifically an acyclic monomer ispositioned in position 5-10 in the guide (antisense) strand of an siRNAduplex, wherein the position is counted from the 5′end of theoligonucleotide.

In another embodiment, the RNA duplex is capable of mediatingtranslational repression or degradation of target mRNA while inducingreduced off-target effects when specifically an acyclic monomer ispositioned in position 6-8 in the guide (antisense) strand of an siRNAduplex. Not intended to be bound by theory, it is believed that thereduced binding affinity induced by the presence of the acyclic monomerat these positions that leads to reduced capability of the guide strandto induce microRNA-type effects. I.e. the acyclic monomer, whenpositioned correctly, reduces so-called seed-region binding, which isassumed to be more important for microRNA activity than for siRNAactivity.

In one embodiment, the RNA duplex is capable of mediating RNA targeting,e.g. gene silencing or RNA interference, with increased potency ascompared to an identical RNA duplex with RNA monomers instead of acyclicmonomers. Increased potency may be achieved because of increasedoff-rate of the cleavage products of RISC reaction. The off -rate may beincreased because of decreased binding affinity. Also increasedflexibility of the substrate may increase the rate of hydrolysis.Furthermore the increased flexibility may ease unwinding of the RNAduplex prior to loading of the guide strand into RISC.

In one embodiment, the RNA duplex is capable of mediating translationalrepression or degradation of target mRNA with prolonged potency ascompared to an identical RNA duplex with RNA monomers instead of acyclicmonomers. Prolonged potency may for example be achieved because theoligonucleotides of the RNA duplex and the duplex per se are a poorersubstrate for exo- and endonucleases and thereby the stability of theoligonucleotides and the duplex is increased.

In one embodiment, the RNA duplex is capable of mediating translationalrepression or degradation of target mRNA wherein the RNA duplex hasimproved biostability as compared to an identical RNA duplex with RNAmonomers instead of acyclic monomers.

In yet another embodiment, the RNA duplex is capable of mediatingtranslational repression or degradation of target mRNA wherein the RNAduplex has reduced immune stimulation as compared to an identical RNAduplex with RNA monomers instead of acyclic monomers. One reason forimmune stimulation is interaction with Toll-like receptors thatrecognizes foreign oligonucleotides. Since RNA duplexes of the inventionare non-natural, they will be more difficult to detect by the Toll-likereceptors.

REFERENCES

-   US2003/0108923-   US2005/0234007-   WO2005/073378-   J. Kurreck, Eur. J. Biochem. 2003, 270, 1628-   K. D. Nielsen et al., Bioorg. Med. Chem. 1995, 3, 1493-   H. Thrane et al., Tetrahedron 1995, 51, 10389-   P. Nielsen et al., Bioorg. Med. Chem. 1995, 3, 19-   Nawrot and Sipa, Curr. Topics Med. Chem. 2006, 6, 913-925-   F. Eckstein, Oligonucleotides and Analogues, IRL Press, Oxford    University Press, 1991-   M. Petersen and J. Wengel, Trends Biotechnol. 2003, 21, 74-81-   Pfundheller, Sørensen, Lomholt, Johansen, Koch and Wengel, J.    “Locked Nucleic Acid Synthesis”, Methods Mol. Biol. 2004, vol. 288    (Oligonucleotide Synthesis), 127-145., P. Herdewijn, Ed., Humana    Press Inc.-   Furniss, Hannaford, Smith and Tatchell, Vogel's Textbook of Organic    Chemistry, 1989, John Wiley & Sons-   Bryld, Højland and Wengel, Chem. Commun. 2004, 1064-   Mokhir, Tetzlaff, Herzberger, Mosbacher and Richart, J. Comb. Chem.    2001, 3, 374-   Mangos M M, Min K L, Viazovkina E, Galarneau A, Elzagheid M I,    Parniak M A, Damha M J., J Am Chem Soc. 2003 January 22;    125(3):654-61

EXPERIMENTAL PROCEDURES AND EXAMPLES Example 1 Synthesis of the RNAComplexes of the Invention

Procedures for preparation of the phosphoramidite building blocks forautomated DNA/RNA synthesis of the hydroxymethyl substituted monomers ofthe RNA complexes of the invention have been reported [thyminederivatives; K. D. Nielsen et al., Bioorg. Med. Chem. 1995, 3, 1493; H.Thrane et al., Tetrahedron 1995, 51, 10389; P. Nielsen et al., Bioorg.Med. Chem. 1995, 3, 19]. Please see Example 11 for disclosure ofprocedures for preparation of example phosphoramidite derivatives ofadenine, guanine, cytosine and uracil.

The incorporation of these hydroxymethyl substituted monomers into theRNA complexes of the invention follows standard methods for a) RNAsynthesis on an automated RNA synthesizer, b) RNA work-up, c) RNApurification and d) RNA isolation [F. Eckstein, Oligonucleotides andAnalogues, IRL Press, Oxford University Press, 1991]. This demonstratesthat hydroxymethyl substituted RNA oligonucleotides (=RNA strands) andRNA complexes can be synthesised using known phosphoramidite derivativesusing the standard techniques for RNA synthesis.

LNA is an oligonucleotide containing one or more2′-O,4′-C-methylene-linked ribonucleotides (LNA nucleotides) [M.Petersen and J. Wengel, Trends Biotechnol. 2003, 21, 74-81].LNA-modified siRNA is an siRNA construct containing one or more LNAmonomers. Known methods have been used to incorporate LNA nucleotidesinto the RNA complexes to the invention by use of the commerciallyavailable LNA phosphoramidites [Pfundheller, Sørensen, Lomholt,Johansen, Koch and Wengel, J. “Locked Nucleic Acid Synthesis”, MethodsMol. Biol. 2004, vol. 288 (Oligonucleotide Synthesis), 127-145, P.Herdewijn, Ed., Humana Press Inc.]

Hydroxymethyl substituted siRNA (“hydroxymethyl substituted smallinterfering RNA) is an siRNA construct containing one or morehydroxymethyl substituted nucleotide monomer (see FIG. 1 for structuresof the hydroxymethyl substituted nucleotide monomer). The monomersexemplified are shown below:

Oligonucleotides—Selected Antisense Strands in siRNA Constructs:

RNA native 5′-ACU UGU GGC CGU UUA CGU CGC U JW1103 5′-ACU UGU GGC CGUUUA CGU C G ^(L) C ^(MeL)  U JW1186 5′-ACU UG T  GGC CGU UUA CGU C G^(L) C ^(MeL)  U JW1187 5′-AC T  UG T  GGC CGU U T A CG T  C G ^(L) C^(MeL)  U W123 5′-ACU UG X  GGC CGU UUA CGU C G ^(L) C ^(MeL)  U W1245′-AC X  UGU GGC CGU UUA CGU C G ^(L) C ^(MeL)  U W125 5′-ACU UGU GGCCGU UUA CG X  C G ^(L) C ^(MeL)  U W126 5′-ACU UGU GGC CG X  UUA CGU C G^(L) C ^(MeL)  U W127 5′-ACU UGU GGC CGU UUA CGT CG X  U W128 5′-AC X UGU GGC CGU UUA CGT CG X  UOligonucleotides—Selected Sense Strands in siRNA Constructs:

RNA 5′-GAC GUA AAC GGC CAC AAG UUC U native JW1104 5′-GAC GUA AAC GGCCAC AAG U T ^(L) C ^(MeL)  U JW1106 5′-GA C ^(MeL)  GUA AA C ^(MeL)  GGCCA C ^(MeL)  AAG U T ^(L) C ^(MeL)  U JW1188 5′-GAC GUA AAC GGC CAC AAGTT C W043 5′-GAC GUA AAC GGC CAC AAG U TC  U W044 5′-GA C  G T A AA C GGC C A C  AAG U TC  U JW1189 5′-GAC G T A AAC GGC CAC AAG TT C W1295′-GAC G X A AAC GGC CAC AAG U T ^(L) C ^(MeL)  U W130 5′-GAC G X A AACX GGC CAC AAG U T ^(L) C ^(MeL)  U W131 5′-GAC GUA AAC GGC CAC AAG UU X U W132 5′-GAC G X A AAC GGC CAC AAG UU X  U

Other hydroxymethyl substituted RNA stands that have been synthesised:

5′-ACU UGU GGC CGU UUA CGU C G C  U 5′-GA C  G T A AA C  G 5′-GC C A C AAG U TC  U

-   -   “L” in superscript indicates that the residue is an LNA        nucleotide.    -   “MeL” in superscript indicates that the residue is an LNA        nucleotide with a 5-methylcytosine base.    -   T in bold face underlined is a hydroxymethyl substituted        nucleotide monomer. In this example it is the thymin-1-yl        derivative of C4′-branched-RNA Monomer C (see FIG. 1).    -   C in bold face underlined is a hydroxymethyl substituted        nucleotide monomer. In this example it is the        5-methylcytosin-1-yl derivative of C4′-branched-RNA Monomer C        (see FIG. 1).    -   X in bold face underlined is a hydroxymethyl substituted acyclic        nucleotide monomer. In the sequences above it is the uracil-1-yl        derivative of 2′,3′-seco-RNA Monomer D (see FIG. 1). In other        examples and figures are other bases variants than uracil        included.

See Examples 9 and 10 for further sequences studied.

Cellular studies (lung cancer cell line expressing EGFP) have beenperformed. As examples to illustrate the invention are used siRNAduplexes containing two or three nucleotide overhangs. This exampledesign is only an illustration and many other constructs are included inthe invention and works similarly. Thus are, for example, blunt endedsiRNA duplexes, shorter or longer siRNA duplexes than the onesexemplified, and single stranded antisense strands included. Likewiseare included RNA complexes comprising an antisense strand and adiscontinued passenger strand (the “passenger strand” can also be calledthe “sense strand”).

Example 2 Annealing and Transfection Procedure for siRNA Complexes ofthe Invention

Cells were plated in 6-well plates and grown on to 40-60% confluence.Immediately before transfection, the cells were re-plated in 1 ml ofcomplete growth media per well. Sense and antisense strands where mixedin annealing buffer (10 mM Tris-HCl, pH 7.3, 50 mM NaCl) at 20 μMconcentration of each and were incubated at 95° C. for 1 min and at 1 hat 37° C. Per well in a 6-well plate, the following solution wasprepared: 4 μl of TranslT-TKO in 150 μl serum free RPMI media. AnnealedsiRNA complex was added, mixed carefully, incubated for 20 min at RT,and poured over the cells. The final RNA complex concentration was 50nM. After 24 h incubation at 37° C., the media was changed and the cellswere incubated for another 24 h at 37° C. The cells were removed bytrypsination and split into half for subsequent RNA and flow analysis.

As gene silencing is achieved (see below), it is demonstrated that theRNA complexes of the invention containing hydroxylmethyl substitutedmonomers can penetrate a cell membrane under standard transfectionconditions.

Example 3 Gene Silencing

Procedure for mRNA and protein quantification. Expression of eGFPprotein was analysed by flow cytometric analysis. Western blotting wasperformed as follows: Cells were washed twice in PBS and an equal amountof cells were lysed in 2 × SDS sample buffer [4% Sodium Dodecyl-Sulphate(SDS), 20% glycerol, 125 mM Tris/HCl pH 6.8, 0.01 mg/ml Bromphenol Blue,10% 2-mercaptoethanol] at 90° C. for 2×10 min separated by gentlepippeting. Proteins were separated in an 8% SDS-acrylamide gel, andelectro-blotted overnight onto a PVDF membrane (Immobilon). The filterwas blocked for 1 h with PBS containing 10% w/v milk. EGFP protein wasdetected using a 1:1000 dilution of a rabbit polyclonal EGFP antibody(Santa Cruz Biotechnology). The mouse hnRNP C1 antibody was a gift fromSeraphin Pinol-Roma. A horse radish peroxidase (hrp) conjugatedsecondary antibody (DAKO) was used with the ECL reagent (AmershamBiosciences) for visualization. eGFP mRNA was analysed by Northernblotting according to standard procedures.

The following is a list with results from gene silencing experimentsconducted at 50 mM siRNA complex concentration. The results are given inpercentages relative to the gene expression level obtained with amis-matched control siRNA duplex (set at 100%):

Entry Sense/Antisense Mean GFP EGFP mRNA 1 RNA/RNA 13% 16% 2JW1104/JW1103 13% 28% 3 JW1188/JW1103 7% 13% 4 JW1189/JW1103 6% 15% 5W043/JW1103 ~13% 6 W044/JW1103 ~19% 7 JW1104/JW1186 22% 31% 8JW1104/JW1187 62% 90% 9 W131/W127 27% 10 W132/W128 86% 11 W131/W128 68%12 W132/W127 47% 13 W129/JW1103 36% 14 W130/JW1103 39% 15 JW1106/W12324% 16 JW1106/W127 51% 17 JW1106/W125 34% 18 JW1106/W126 22%

Entry 1 shows that the unmodified siRNA complex is efficiently silencingthe GFP gene.

Entry 2 shows that an LNA-modified siRNA complex is efficientlysilencing the GFP gene. This construct has two LNA modifications towardsthe 3′-ends of the two RNA strands.

In the example gene silencing experiments of entries 3-8 are studied ansiRNA complex containing C4′-branced-RNA hydroxymethyl substitutedmonomers of structure T/C (FIG. 3).

Entry 3 shows that an siRNA complex of the invention having ahydroxymethyl substituted monomer at positions 2 and 3 from the 3′-endof the sense strand is highly functional in silencing the GFP gene.

In the example of entry 3 and in the examples of entries 4, 5, 6, 13 and14 is the antisense strand as an example an LNA-modified RNA strand, butan unmodified RNA antisense strand or a fully or partially modified RNAantisense strand would also be functional. The results obtained showthat alternatively modified monomers like LNA monomers are fullycompatible with the hydroxymethyl substituted monomers of the invention.

Entry 4 confirms that an siRNA complex of the invention havinghydroxymethyl substituted monomers T/C in the sense strand is highlyefficient in mediating gene silencing.

Entries 5 and 6 confirm that an siRNA complex of the invention havinghydroxymethyl substituted monomers T/C in the sense strand is highlyefficient in mediating gene silencing.

The results show that very efficient gene silencing is achieved withsiRNA complexes of the invention having hydroxymethyl substitutedmonomers incorporated into the sense strand. The data show thesurprising finding that in general even improved gene silencing isachieved with these RNA complexes of the invention when compared withthe gene silencing achieved with unmodified siRNA or LNA-modified siRNA.Furthermore, silencing is efficient even with an RNA complex comprisinga sense RNA strand with several hydroxymethyl substituted monomers inthe central duplex forming core region (entry, W044 as example).

Entries 7 and 8 reveal that an siRNA complex of the invention havinghydroxymethyl substituted monomers T/C in the antisense strand of thecomplex is able to mediate gene silencing. It seems that the morehydroxymethyl substituted monomers T/C that is incorporated into theantisense strand the lower the gene silencing activity.

An LNA-modified sense strand is used as an example in the examples ofentries 7, 8, 15, 16, 17 and 18, but an unmodified RNA sense strand or afully or partially modified RNA sense strand would also be functional.The results obtained show that alternatively modified monomers like LNAmonomers are fully compatible with the hydroxymethyl substitutedmonomers of the invention.

In the example gene silencing experiments of entries 9-18 are studied ansiRNA complex of the invention containing 2′,3′-seco-RNA hydroxymethylsubstituted monomer of structure X shown above in FIG. 3.

Entry 9 demonstrate that an siRNA complex of the invention having onehydroxymethyl substituted monomer X in each of the two RNA strands(towards the 3′-end of the two strands) mediate very efficient genesilencing to a level comparable to that of unmodified siRNA. This issurprising taking the non-cyclic nature of the ribose unit of thehydroxymethyl substituted monomer X into consideration.

With an additional X monomer in each of the two strands of the RNAcomplex, gene silencing efficiency is inefficient (entry 10).

Entry 11 reveals together with entry 10 that incorporation of ahydroxymethyl substituted monomer X close to the 5′-end of the antisensestrand reduces the gene silencing efficiency of an siRNA complex.

Entry 12 reveals that incorporation of a hydroxymethyl substitutedmonomer X close to the 5′-end of the sense strand reduces the genesilencing efficiency of an siRNA complex when another monomer X isincorporated into the 3′-end of the sense strand.

Entries 13 and 14 confirm that incorporation of a hydroxymethylsubstituted monomer X close to the 5′-end of the sense strand reducesthe gene silencing efficiency of an siRNA complex. The results indicatethat incorporation of a hydroxymethyl substituted monomer X in thecentral part of a sense strand of an siRNA construct is neitherimproving nor reducing gene silencing activity.

Entries 15-18 display results from gene silencing experiments with siRNAcomplexes of the invention comprising an LNA-modified RNA sense strandand antisense strands having one hydroxymethyl substituted monomer X.

The results show that siRNA complexes of the invention that containhydroxymethyl substituted monomers X in the central region of theantisense strand, e.g. W126 and W123, mediate very efficient genesilencing. It is surprising that W127, which together with W131 mediatesvery efficient gene silencing, only induces moderate gene silencing withthe LNA-modified RNA sense strand JW1106. This underlines the surprisingaspect of the observation (entry 9) that an siRNA complex of theinvention having one hydroxymethyl substituted monomer X in each of thetwo RNA strands (towards the 3′-ends of the two strands) mediate veryefficient gene silencing to a level comparable to that of unmodifiedsiRNA.

Results similar to the ones described above can be obtained with the RNAcomplexes of the invention containing hydroxymethyl substituted monomershaving other nucleobases than uracil, thymine or 5-methylcytosine. Forexample can comparable gene silencing activities using similar protecolsbe obtained for the RNA complexes of the invention containinghydroxymethyl substituted monomers having adenine, cytosine or guanineas nucleobases.

Example 4 Immune Stimulation

Because the RNA complexes of the invention containing hydroxymethylsubstituted monomers are chemically modified relative to thecorresponding unmodified RNA complexes, they will display less immunestimulatory activity than the corresponding unmodified RNA complexes.

Example 5 Off-target Effects

Because the RNA complexes of the invention containing hydroxymethylsubstituted monomers can be modulated such thatantisense-strand-modified siRNA complexes are inactive, gene silencingwith less off-target effects is made possible by the invention. The keyis to modify the sense strands with hydroxymethyl substituted monomerssuch that the sense strand cannot function as the antisense strand. Thiscan e.g. be achieved by incorporating a hydroxymethyl substitutedmonomer towards the 5′-end of the sense strand.

With the acyclic monomer X reduced off-target effects can be achieved byincorporating monomer X in the antisense strand, most preferable aroundpositions 6-8 from the 5′-end of the antisense strand, for example atposition 7 from the 5′-end of the antisense strand.

Example 6 Synthesis of the RNA Complexes of the Invention ContainingFunctionalised and Conjugated hydroxymethyl monomers

The hydroxymethyl substituent of the hydroxymethyl substituted monomersof the invention is functionalised by a conjugating group. A conjugatinggroup is herein defined as a group that modulates, expands or improvesthe chemical, biophysical or biological properties of an RNA complex ofthe invention. Such groups may be useful for modulating cellulardistribution, organ distribution, tissue distribution, meltingtemperatures, target affinities, biostability, signalling ofhybridization etc.

Known methods can be used to convert a hydroxymethyl substituent into avariety of chemical derivatives [Furniss, Hannaford, Smith and Tatchell,Vogel's Textbook of Organic Chemistry, 1989, John Wiley & Sons]. Thiscan be achieved at the nucleoside level, i.e. before conversion into aphosphoramidite derivative useful for automated RNA synthesis on anautomated DNA synthesiser. After conversion of the hydroxymethyl groupinto a useful derivative, the phosphoramidite derivative needed forautomated RNA synthesis is synthesised using standard methods, andincorporation of the derivatised or conjugated monomers into RNAoligonucleotides (strands) is subsequently achieved using standardmethods (see Example 1).

Conjugation via an ether linkage. The hydroxymethyl substituent of thehydroxymethyl substituted monomers of the invention is functionalised byan ether linkage between a conjugating group and the methylene group ofthe hydroxymethyl substituent by a nucleophilic substitution reaction.This reaction involves conversion of the hydroxy group of thehydroxymethyl substituent into a good leaving group by e.g. mesylationor transformation into a halide, and subsequent nucleophilic attach byan alcohol or an alkoxide derivative.

Conjugation via a thioether linkage. The hydroxymethyl substituent ofthe hydroxymethyl substituted monomers of the invention isfunctionalised by a thioether linkage between a conjugating group andthe methylene group of the hydroxymethyl substituent by a nucleophilicsubstitution reaction. This reaction involves conversion of the hydroxygroup of the hydroxymethyl substituent into a good leaving group by e.g.mesylation or transformation into a halide, and subsequent nucleophilicattach by an alkylthiol or thioalkoxide derivative. If the nucleophilealternatively is SH⁻, protection by e.g. acetylation leads to aphosphoramidite derivative that is useful for introduction of mercapto(SH) groups into the RNA complexes of the invention. As an alternativeprocedure to introduce a mearcapto functionality into the RNA complexescan conjugation with a disulfide containing moiety be used. Afterreduction of the disulfide containing RNA complex is the mercapto groupfunctionalised RNA complex obtained.

Derivatisation into an aminomethyl group. The hydroxymethyl substituentof the hydroxymethyl substituted monomers of the invention can beconverted into an aminomethyl group. This reaction involves conversionof the hydroxy group of the hydroxymethyl substituent into a goodleaving group by e.g. mesylation or transformation into a halide, andsubsequent nucleophilic attach by ammonia or a protected aminederivative (like e.g. phthalimide) that subsequently is deprotected (forexample after RNA synthesis) to give the desired amino derivative. Atrifluoroacetyl or Fmoc protecting group are other options foramino-protection during automated RNA synthesis, with liberation of afree amino group after standard oligonucleotide deprotection.

Conjugation via an amide linkage. The hydroxymethyl substituent of thehydroxymethyl substituted monomers of the invention is acting as ahandle for attachment of amide-linked conjugating groups. This involvesconversion of the hydroxy unit of the hydroxymethyl substituent into anamine unit, for example as described above, and further derivatisationof this amino group by e.g. a conjugating group via amide bond formationusing known methods. This may take place before RNA synthesis or afterRNA synthesis using methods known to a person skilled in the art [Bryld,Højland and Wengel, Chem. Commun. 2004, 1064; Mokhir, Tetzlaff,Herzberger, Mosbacher and Richart, J. Comb. Chem. 2001, 3, 374].

Conjugation via an amino linkage. The hydroxymethyl substituent of thehydroxymethyl substituted monomers of the invention is also acting as ahandle for attachment of amino-linked conjugating groups. This involvesconversion of the hydroxy unit of the hydroxymethyl substituent into anamine unit, for example as described above, and further derivatisationof this amino group by a conjugating group containing e.g. an aldehydefunctionality by a reductive amination reaction which is a knownreaction [Furniss, Hannaford, Smith and Tatchell, Vogel's Textbook ofOrganic Chemistry, 1989, John Wiley & Sons]. This may take place beforeRNA synthesis or after RNA synthesis.

Conjugation via a piperazino group or a linear diamino alkyl group. Apiperazino group or a linear diamino alkyl group is also used forderivatisation by performing reactions as described [Bryld, Højland andWengel, Chem. Commun. 2004, 1064-5]. These groups will be useful, asother conjugating groups can be attached at e.g. the distal nitrogenatom of a piperazino group (see FIG. 2, Monomer J) by e.g. amide bondformation or by a reductive amination reaction, either before or afterRNA oligonucleotide synthesis [Bryld, Højland and Wengel, Chem. Commun.2004, 1064; Mokhir, Tetzlaff, Herzberger, Mosbacher and Richart, J.Comb. Chem. 2001, 3, 374]. This way can e.g. cholesteryl or fatty acidunits be linked to the RNA complexes of the invention via apiperazino-methyl substituent.

Using these procedures the RNA complexes of the invention can beprepared containing e.g. cholesteryl units, alkyl units, fatty acidunits, polyamine derivatives, thio derivatives, amino acids,polypeptides, monosaccharide derivatives, polysaccharide derivatives orfluorophores, all connected to the RNA complexes of the invention viathe methylene group of the hydroxymethyl substituent. The groups listedhere are only examples of groups that can be attached using theprocedures exemplified above. See FIG. 2 for structural examples of theconjugated monomers.

Example 7 Gene Silencing by the RNA Complexes of the InventionContaining Functionalised and Conjugated hydroxymethyl monomers

Gene silencing is efficient with the RNA complexes of the inventioncontaining the functionalised and conjugated hydroxymethyl monomers (seee.g. FIG. 2 or Example 6).

Efficient gene silencing is achieved when these functionalised andconjugated hydroxymethyl monomers are positioned at or close to the3′-ends of the two strands of an siRNA complex.

Efficient gene silencing is furthermore achieved when thesefunctionalised and conjugated hydroxymethyl monomers are positioned ator close to the 3′-end and the 5′-end of the sense strand of an siRNAcomplex.

Efficient gene silencing is in particular achieved when thesefunctionalised and conjugated hydroxymethyl monomers are positioned ator close to the 3′-end of the sense strand of an siRNA complex.

Efficient gene silencing is furthermore achieved when thesefunctionalised and conjugated hydroxymethyl monomers are positioned in asingle stranded antisense RNA oligonucleotide.

Modulation of pharmacokinetic properties is achieved together withefficient gene silencing when the group (R in FIG. 2) of an RNA complexof the invention is a cholesteryl derivative. This leads for example toimproved tissue distribution and cellular uptake, and also increasedbiostability.

Modulation of pharmacokinetic properties is achieved together withefficient gene silencing when the group (R in FIG. 2) of an RNA complexof the invention is a thio derivative. This leads to improvedcirculation time in a human body, i.e. reduced clearance via thekidneys.

Modulation of pharmacokinetic properties is achieved together withefficient gene silencing when the group (R in FIG. 2) of an RNA complexof the invention contains an amino group. This leads to improved tissuedistribution.

Example 8 Biostability of the Hydroxymethyl-substituted RNA Complexes

Experimental procedure for the stability assay. The hydroxymethylsubstituted RNA complexes were incubated at 37° C. in 10% fetal bovineserum (Gibco) diluted in D-MEM (Gibco). Samples of 5 μl were collectedat indicated time points and immediately frozen on dry ice in 15 μl1.33×TBE/10% glycerol loading buffer and subjected to non-denaturingPAGE on a 15% gel. RNAs were visualised with SYBR gold (Invitrogen).

Such experiments show that the stability of thehydroxymethyl-substituted RNA complexes display improved stability inbiological media relative to the native (or “unmodified”) control RNAcomplexes. Thus the hydroxymethyl-substituted siRNAs are significantlymore stable in 10% serum than ordinary siRNA. It can thus be envisionedthat only a very small decline in hydroxymethyl-substituted siRNA sizeis observed over a more than one hour long incubation period. Weconclude that the RNA complexes of the invention containinghydroxymethyl substituted monomers are very stable in cells, in animalsand in humans, and that this characteristic is contributing to theirvery efficient gene silencing properties. Because of this pronouncedbiostability, the RNA complexes of the invention containinghydroxymethyl substituted monomers display gene silencing for a longerperiod of time than their unmodified counterparts.

Example 9 A Series of Gene Silencing Experiments Demonstrating theStrong Potential of monomers of Structure D for Gene Silencing

Using procedures described in the prior experiments were gene silencingstudies conducted using siRNA duplexes of the sequences describedearlier as well as of additional sequences (see FIGS. 4-9 for thesequences included in the studies of this example). These experimentsincluded sequences containing one or more incorporations of monomer X(see Example 1 for description of monomer X). Monomer X is used hereinonly as an example structure and similar results are predicted forderivatives like e.g. monomer E, monomer F, monomer G, monomer I andmonomer J (see FIG. 1 and FIG. 2). The bold and underlined monomers witha superscript L are LNA monomers. In this example, including FIGS. 4-9are C ^(L)=C^(MeL) =5-methylcytosin-1-yl LNA monomer.

The experiments for which the results are depicted in FIG. 4 all involvea sense strand that has two incorporations of monomer X (W130). It isshown that:

-   -   it is possible to design a siRNA duplex composed of strands        containing both hydroxymethyl-substituted and LNA monomers that        display gene silencing functionality;    -   it is possible to design a siRNA duplex composed of strand        having a mismatched monomer X in the sense strand that display        gene silencing functionality;    -   the full RNA antisense strand (except for two LNA monomers        toward the 3′-end) is well tolerated;    -   a single X monomer is well tolerated in the antisense strand        (W123, W125 or W126);    -   several X monomers are tolerated but less efficient gene        silencing is observed with W186 and W187 than with W123, W125 or        W126 as antisense strand;    -   significant gene silencing activity is seen with W188 though        this antisense strand contains six LNA monomers which shows that        a monomer X positioned centrally in an antisense strand is able        to improve the gene silencing relative to the situation in which        monomer X is substituted with the corresponding RNA monomer.

The experiments for which the results are depicted in FIG. 5 all involvea sense strand that has one monomer X positioned toward the 3′-end ofthe strand (W131). It is shown that:

-   -   the full RNA antisense strand (except for two LNA monomers        toward the 3′-end) is well tolerated;    -   a single X monomer may be well tolerated in the antisense strand        (W123);    -   a single X monomer might lead to as good or even improved gene        silencing relative to the unmodified control (siRNA-EGFP) (W125        or W126);    -   several X monomers are rather well tolerated (W186, W187 or W281        as antisense strand);    -   significant gene silencing activity is seen with W188 though        this antisense strand contains six LNA monomers which shows that        a monomer X positioned centrally in an antisense strand is able        to improve the gene silencing relative to the situation in which        monomer X is substituted with the corresponding RNA monomer;    -   significant gene silencing is observed with several        substitutions of monomer X in the antisense strand in a        situation without the co-presence of LNA monomers (W281).

The experiments for which the results are depicted in FIG. 6 all involvea sense strand that has three X monomers dispersed along the sensestrand (W282). It is shown that:

-   -   the full RNA antisense strand (except for two LNA monomers        toward the 3′-end) is well tolerated;    -   a single X monomer may be well tolerated in the antisense        strand, most so apparently toward the 3′-end for which as good        or even improved gene silencing relative to the unmodified        control (siRNA-EGFP) was observed (W123, W125, W126);    -   several X monomers are rather well tolerated (W186, W187 or W281        as antisense strand);    -   significant gene silencing activity is seen with W188 though        this antisense strand contains six LNA monomers which shows that        a monomer X positioned centrally in an antisense strand is able        to improve the gene silencing relative to the situation in which        monomer X is substituted with the corresponding RNA monomer, and        that also when the sense strand contains several X monomers;    -   gene silencing is observed with several substitutions of monomer        X in the antisense strand also in a situation without the        co-presence of LNA monomers (W281).

The experiments for which the results are depicted in FIG. 7 all involvea sense strand without monomer X (W194). It is shown that:

-   -   a single X monomer may be well tolerated in the antisense strand        (W123);    -   a single X monomer might lead to as good or even improved gene        silencing relative to the unmodified control (siRNA-EGFP) (W125        or W126);    -   several X monomers are rather well tolerated (W186, W187 or W281        as antisense strand);    -   significant gene silencing activity is seen with W188 though        this antisense strand contains six LNA monomers which shows that        a monomer X positioned centrally in an antisense strand is able        to improve the gene silencing relative to the situation in which        monomer X is substituted with the corresponding RNA monomer;    -   significant gene silencing is observed with several        substitutions of monomer X in the antisense strand also in a        situation without the co-presence of LNA monomers (W281).

The experiments for which the results are depicted in FIG. 8 all involvea sense strand without monomer X (W181) but with four LNA monomersincorporated in the duplex forming segment (plus two LNA monomers in the3′-end). It is shown that:

-   -   a single X monomer is well tolerated in the antisense strand        (W123, W125 or W126);    -   several X monomers are rather well tolerated (W186, W187 or W281        as antisense strand);    -   significant gene silencing activity is seen with W188 though        this antisense strand contains six LNA monomers which shows that        a monomer X positioned centrally in an antisense strand is able        to improve the gene silencing relative to the situation in which        monomer X is substituted with the corresponding RNA monomer;    -   significant gene silencing is observed with several        substitutions of monomer X in the antisense strand also in a        situation without the co-presence of LNA monomers (W281).

The experiments for which the results are depicted in FIG. 9 all involvea sense strand that has one monomer X positioned toward the 5′-end ofthe strand (W129). It is shown that:

-   -   the full RNA antisense strand (except for two LNA monomers        toward the 3′-end) is well tolerated;    -   a single X monomer may be well tolerated in the antisense strand        and might lead to improved gene silencing relative to the        unmodified control (siRNA-EGFP) (W123, W125 or W126);    -   several X monomers are rather well tolerated (W186, W187 or W281        as antisense strand);    -   significant gene silencing activity is seen with W188 though        this antisense strand contains six LNA monomers which shows that        a monomer X positioned centrally in an antisense strand is able        to improve the gene silencing relative to the situation in which        monomer X is substituted with the corresponding RNA monomer;    -   significant gene silencing is observed with several        substitutions of monomer X in the antisense strand in a        situation without the co-presence of LNA monomers (W281).

The data shown below indicate that hydroxymethyl-substituted monomersare compatible with the sisiRNA approach. As an example, the use of thetri-molecular combination of the W123 antisense strand+(W004+W005) sensestrands leads to efficient gene silencing (i.e., “siRNA effect” has alow value, in the case of the siRNA effect is 0.24) which shows thatmonomer X may be positioned in the antisense strand of sisiRNA complexes(compare to Control; read out 1.0). Data for unmodified siRNA is alsoshown. The design of the antisense strand is important, as thecombination of the W186 antisense strand+(W004+W005) sense strands isunable to induce a gene silencing effect. This shows that the number ofhydroxymethyl-substituted monomers (e.g. Monomer D) should be low, andmost favourably restricted to one monomer (besides optionalhydroxymethyl-substituted monomers in the overhang of the antisensestrand). Other RNA complexes included in the study depicted in FIG. 9are equally efficient with respect to gene silencing.

Antisense strand siRNA effect W123 0.24 W125 0.26 W126 0.23 W186 1.12SiRNA 0.11 Control 1.0

Example 10 Seed Modifications Reduce Off-target Effects

Using antisense strand W124, and W207 (5′-GAC GUA AAC GGC CAC AAG UT^(L)C^(MeL) ) (SEQ ID NO:) as sense strand in an siRNA duplex at 50 nMconcentration, we have shown that the monomer X when present in theso-called seed-region of the antisense strand has a selectivityenhancing effect which will lead to less off-target effects. Theexperimental setup thus allowed discrimination between siRNA effect(gene silencing with strand cleavage) and miRNA effect (translationalrepression; plasmid-based off-target sensor having four target regionscomposed of only seed region matching). Using the combination above, thesiRNA effect was as for the unmodified siRNA control whereas the miRNAeffect was significantly reduced. A similar effect was obtained for thesiDharma (a commercial product having a 2′-O-Me-RNA monomer incorporatedin position no. 2 from the 5′-end of the antisense strand). As statedabove this shows that a hydroxymethyl-substituted monomer (e.g. MonomerD) present in the so-called seed region of the antisense strand leads toa favourable effect (i.e., reduced or elimination of off-target effect).Thus, a method for reducing or eliminating off-target effect of an RNAcomplex, the method comprising incorporating one or morehydroxymethyl-substituted monomers (e.g. Monomer D) in an RNA complex orpreparing an RNA complex containing one or morehydroxymethyl-substituted monomers (e.g. Monomer D). See table below forthe results for gene silencing (i.e., “siRNA effect”) and off-targeteffect (i.e., “miRNA effect). These data indicate that an RNA complexcontaining one or more hydroxymethyl-substituted monomers (e.g., MonomerD) reduce the expression of the target while minimizing off-targeteffect.

Antisense strand siRNA effect miRNA effect W124 0.12 0.38 W125 0.04 0.19SiRNA 0.06 0.15 siDharma 0.08 0.37 Control 1.0 1.0

For further seed walk studies, we have prepared the following sequencescomposed of RNA monomers (rA, rC, rG and rU) and hydroxymethyl-modifiedmonomers (Monomer D; labeled sA, sC, sG and sU for the adenin-9-yl,cytosin-1-yl, guanin-9-yl and uracil-1-yl derivatives, respectively).Monomer X represents Monomer D with a nucleobase:

The numbering of the first nine oligonucleotides shown below are asfollows (from no. 1 from the top—no. 9):

W313; W314; W315; W316; W317; W123; W318; W319 and W320 sA rC rU rU rGrU rG rG rC rC rG rU rU rU rA rC rG rU rC lG lC rU rA sC rU rU rG rU rGrG rC rC rG rU rU rU rA rC rG rU rC lG lC rU rA rC sU rU rG rU rG rG rCrC rG rU rU rU rA rC rG rU rC lG lC rU rA rC rU sU rG rU rG rG rC rC rGrU rU rU rA rC rG rU rC lG lC rU rA rC rU rU sG rU rG rG rC rC rG rU rUrU rA rC rG rU rC lG lC rU rA rC rU rU rG sU rG rG rC rC rG rU rU rU rArC rG rU rC lG lC rU rA rC rU rU rG rU sG rG rC rC rG rU rU rU rA rC rGrU rC lG lC rU rA rC rU rU rG rU rG sG rC rC rG rU rU rU rA rC rG rU rClG lC rU rA rC rU rU rG rU rG rG sC rC rG rU rU rU rA rC rG rU rC lG lCrU sA rC rU rU rG rU rG rG rC rC rG rU rU rU rA rC rG rU rC rG sU rU rAsC rU rU rG rU rG rG rC rC rG rU rU rU rA rC rG rU rC rG sU rU rA rC sUrU rG rU rG rG rC rC rG rU rU rU rA rC rG rU rC rG sU rU rA rC rU sU rGrU rG rG rC rC rG rU rU rU rA rC rG rU rC rG sU rU rA rC rU rU sG rU rGrG rC rC rG rU rU rU rA rC rG rU rC rG sU rU rA rC rU rU rG sU rG rG rCrC rG rU rU rU rA rC rG rU rC rG sU rU rA rC rU rU rG rU sG rG rC rC rGrU rU rU rA rC rG rU rC rG sU rU rA rC rU rU rG rU rG sG rC rC rG rU rUrU rA rC rG rU rC rG sU rU rA rC rU rU rG rU rG rG sC rC rG rU rU rU rArC rG rU rC rG sU rU

By using similar experimental techniques as described in previousexamples with oligomers W313; W314; W315; W316; W317; W123; W318; W319and W320 as antisense strand and oligomer JW1104 as sense strand in genesilencing experiments is was shown that the potency of the siRNAconstructs containing the hydroxymethyl-substituted monomer D can beimproved relative to unmodified siRNA or a commercial chemicallymodified siRNA product (Dharma) having a 2′-O-Me-RNA monomer in positionno. 2 from the 5′-end of the antisense strand.). Further, siRNAcomplexes containing 2′,3′-seco-RNA Monomer D showing that this monomercan be incorporated in siRNA constructs such that off-target effects(miRNA effects) are reduced relative to the reference unmodified siRNA(SiRNA)

The results for 1 nM, 10 nM and 100 nM RNA complexes are depicted intabular form below (Control; data adjusted to 1.0). Gene silencing viasiRNA and miRNA (microRNA) effects were studied at variousconcentrations of the different siRNA duplexes as indicated. Similarresults are expected from strands like the nine strands shown abovecontaining exclusively RNA and acyclic 2′,3′-seco-RNA monomers.

Hydroxymethyl-modified monomer D as antisense strand modificationreduces off-target effects and increases potency of gene silencing.

SiRNA effect miRNA effect 1 nm 10 nm 100 nm 1 nm 10 nm W313 0.45 0.760.81 0.52 0.67 W314 0.58 0.96 0.88 0.72 0.64 W315 0.48 0.82 0.85 0.620.64 W316 0.46 0.75 0.78 0.85 0.97 W317 0.26 0.32 0.92 0.39 0.52 W3180.17 0.18 0.45 0.46 0.62 W319 0.34 0.50 0.66 0.23 0.29 W320 0.87 0.960.97 0.13 0.12 Dharma 0.52 0.47 0.89 0.37 0.40 SiRNA 0.23 0.24 0.54 0.120.14 Control 1.0 1.0 1.0 1.0 1.0

The design is important, and the most potent of the above mentionedseries of oligomers for siRNA effect is W318 which has ahydroxymethyl-substituted monomer D at position no. 7 from the 5′-end ofthe antisense strand. W318 also leads to favorably low off target(miRNA) effects relative to unmodified siRNA or a commercial product(Dharma). In general the use of the antisense strands listed abovehaving a hydroxymethyl-substituted monomer D incorporated leads tofavorably low off target (miRNA) effects. Importantly and surprisingly,high potency and low off target effects can simultaneously be realisedusing a construct with an antisense strand containing ahydroxymethyl-substituted monomer D (see table above). In particular thedesign of W318 is favorable showing that a hydroxymethyl-substitutedmonomer D can favorably be incorporated around the boarder of theso-called seed region of the antisense strand, most favorably aroundpositions no. 5-10 from the 5′-end of the antisense strand, like e.g.position no. 7 from the 5′-end of the antisense strand. Additionalincorporations of one or more hydroxymethyl-substituted monomer D can berealised in the two strands.

It can furthermore be note that the effect can be reversed if monomer Xis positioned in the antisense strand around positions 9-16, wherein thepositions are counted from the 5′end. If for example monomer X in theantisense strand is present in position no 9 from the 5′-end of theantisense strand, the antisense strand and the duplex acts as a microRNA(the siRNA effect will be minimal and the microRNA effect much higher).This effect possibly stems from the reduced tendency towards full lengthbinding because of reduced affinity caused by the presence of an acyclichydroxymethyl substituted monomer X (=monomer D).

Example 11 Synthesis of Phosphoramidite Monomeric Building Blocks

The scheme below displays procedures that have been conducted in orderto exemplify synthesis of monomeric amidite (=phosphoramidite) buildingblocks:

Compounds 100 are ribonucleoside starting materials. Compounds 102 arediols prepared by oxidative cleavage reactions followed by reduction.Compounds 103 are O2′-benzoylated derivatives prepared by selectivebenzoylation of the O2′-hydroxy group of compounds 102. Compounds 104are amidites (=phosphoramidites) prepared by O3′-phosphorylation of theO3′-hydroxy group of compounds 103. Below are detailed procedures andcharacterization data included as example procedures.

5′-O-(4,4′-Dimethoxytrityl)-2′,3′-secouridine (102-U)

Nucleoside 100-U (5′-O-(4,4′-Dimethoxytrityl)uridine; 10.35 g, 18.94mmol) was dissolved in dioxane (250 mL) and water (50 mL). NaIO₄ (4.47g, 20.90 mmol) was dissolved in water (50 mL) and added to the dissolvednucleoside. The mixture was stirred for 1 h, during which a whiteprecipitate was formed. Additional dioxane (200 mL) was added and thesuspension was stirred for 15 min, whereupon the suspension was filteredthrough a glass filter and the filter cake was washed with dioxane (100mL). The filtrates were combined, NaBH₄ (797 mg, 21.1 mmol) was addedand the reaction mixture stirred for 30 min. The reaction mixture wasneutralized with a buffer (pyridine:AcOH 1:1, v/v, ˜10 mL). Afterevaporation of the mixture to approximately 150 mL CH₂Cl₂ (100 mL) wasadded and the mixture washed with sat. aq. NaHCO₃ (2×100 mL). Theorganic phase was separated, dried with Na₂SO₄, evaporated to dryness,and the resulting residue was purified by column chromatography (40%acetone in petroleum ether) affording the desired nucleoside 102-U as awhite foam after evaporation of the solvents.

Yield: 8.53 g (82%).

R_(f): 0.2 (10% MeOH in CH₂Cl₂).

¹H NMR (DMSO-d₆): δ 11.34 (br s, NH), 7.62 (d, 1H, J=8.05 Hz, H6),7.45-7.15 (m, 9H, ar), 6.85 (d, 4H, ar), 5.80 (t, 1H, J=6.2 Hz, H1′),5.52 (d, 1H, J=8.05 Hz, H5), 5.12 (t, 1H, J=5.86 Hz, 2′OH), 4.74 (t, 1H,J=5.49 Hz, 3′OH), 3.72 (s, 6H, OCH₃), 3.55-3.47 (m, 3H, H2′/H4′), 3.40(t, 2H, J=5.13 Hz, H3′), 3.01-2.90 (m, 2H, H5′).

¹³C NMR (DMSO-d₆): δ 163.2, 157.9, 151.4, 144.8, 141.1 (C5), 135.4,129.5 (ar), 127.7, 127.5 (ar), 126.5 (ar), 113.0, 101.6 (C6), 85.3, 83.6(C1′), 79.3 (C2′/C4′), 63.5 (C5′), 61.1, 60.5 (C2′/C4′), 54.9 (—OCH₃).

ESI-HiRes (mNa⁺): m/z: 571.1743 calc.: 571.2051.

2′-O-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′,3′-secouridine (103-U)

Nucleoside 102-U (3.01 g, 5.50 mmol) was coevaporated with an. toluene(15 mL). The resulting residue was dissolved in an. DCM (150 mL) alongwith an. Pyr. (4.4 mL) and the mixture was cooled to −70° C. Benzoylchloride (700 μL, 6 mmol) was slowly added to the reaction mixture andstirred for 1 h at −70° C. EtOH (5 mL) was added to the solution andsubsequently allowed to reach rt. The reaction mixture was washed withsat. aq. NaHCO₃ (3×100 mL) and brine (100 mL). The combined aqueousphase was back extracted with CH₂Cl₂ (100 mL). The organic phases werecombined and evaporated. The resulting residue was purified by columnchromatography (3.5% MeOH in DCM) affording the product 103-U as a whitefoam after evaporation of the solvents.

Yield: 3.44 g (79%).

R_(f): 0.3 (5% MeOH in CH₂Cl₂).

¹H NMR (DMSO-d₆): δ11.43 (s, 1H, NH, ex), 7.93-7.87 (m, 2H, ar), 7.80(d, 1H, J=8.05 Hz, H6), 7.70-7.63 (m, 1H), 7.56-7.48 (m, 2H, ar),7.35-7.17 (m, 10H, ar), 6.89-6.81 (m, 4H, ar), 6.20 (t, 1H, J=5.49 Hz,H1′), 5.56 (d, 1H, J=8.05 Hz, H5), 4.83 (t, 1H, OH-3′, ex), 4.58 (dq,2H, H2′), 3.73 (s, 7H, —OCH₃), 3.70-3.62 (m, 1H, H4′), 3.45 (t, 2H,H3′), 3.11-2.96 (m, 2H, H5′).

¹³C NMR (DMSO-d₆): δ 164.92, 162.98, 157.89, 151.00, 144.67, 140.51,135.45, 135.35, 133.54, 129.49, 129.45, 129.13, 129.02, 128.92, 128.74,128.61, 127.65, 127.51, 126.49, 113.16, 113.01, 102.06, 85.35, 80.84,79.57, 71.8, 71.8, 63.4, 60.5, 54.9, 54.8.

ESI-HiRes (mNa⁺): m/z: 675.1949 calc.: 675.2313.

2′-O-Benzoyl-3′-O-(2-cyanoethoxy(diisopropylamino)phosphino)-5′-O-(4,4′-dimethoxytrityl)-2′,3′-secouridine(104-U)

Nucleoside 103-U (679 mg, 1.04 mmol) was coevaporated with DCE (3×6 mL)and dried for 12 h in vacuo. The residue was dissolved in 20% DIPEA inMeCN (6.5 mL) and the mixture was stirred.2-Cyanoethyl-N,N-diisopropylchlorophosphoramidite[P(Cl)(OCH₂CH₂CN)(N(iPr)₂): 0.66 mL, 3.02 mmol] was added to thereaction mixture and stirring was continued for 40 min. The reactionmixture was poured into DCE (10 mL) and washed with sat. aq. NaHCO₃ (10mL) and the aqueous phase was back extracted with DCE (10 mL). Theorganic phases were pooled and evaporated to afford white foam. Thecrude product was purified by column chromatography (0-20% EtOAc intoluene) to give nucleoside 104-U as a white solid material afterevaporation of the solvents.

Yield: 600 mg (68%).

R_(f): 0.6 (50% EtOAc in toluene).

³¹P NMR (MeCN): δ 147.8.

ESI-HiRes (mNa⁺): m/z: 875.2946 calc.: 875.3391.

6-N-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′,3′-secoadenosine (102-A)

6-N-Benzoyl-5′-O-(4,4′-Dimethoxytrityl)adenosine (100-A; 7.02 g, 10.42mmol) was dissolved in dioxane (150 mL) and water (25 mL). NaIO₄ (2.73g, 11.77 mmol) was dissolved in water (25 mL) and added to the dissolvednucleoside. The mixture was stirred for 1 h, during which a whiteprecipitate was formed. Additional dioxane (100 mL) was added and thesuspension was stirred for 15 min, whereupon the suspension was filteredthrough a glass filter and the filter cake washed with dioxane (50 mL).The filtrates were combined, NaBH₄ (435 mg, 11.5 mmol) was added and thereaction mixture stirred for 30 min. Acetone was then added to quenchresidual NaBH₄. The reaction mixture was neutralized with a buffer(pyridine:AcOH 1:1, v/v, ˜10 mL). The reaction mixture was reduced toapproximately 100 mL and CH₂Cl₂ (100 mL) was added and the mixturewashed with sat. aq. NaHCO₃ (2×100 mL). The organic phase was separated,dried with Na₂SO₄, evaporated to dryness, and the resulting residue waspurified by column chromatography using DCM and i-PrOH to give theproduct as a white solid material after evaporation of the solvents.

Yield: 6.07 g (86%). R_(f): 0.22 (5% i-PrOH in DCM).

¹H NMR (DMSO-d₆): δ 11.23 (br s, 1H, N6H), 8.76 (s, 1H, adenin C8/C2),8.68 (s, 1H, adenin C8/C2), 8.07 (d, 2H, J=6.96 Hz, Ar), 7.69-7.50 (m,3H, Ar), 7.25-6.91 (m, 9H, Ar), 6.79 (dd, 4H, Ar), 6.06 (t, 1H, H1′),5.29 (t, 1H, 2′OH), 4.84 (t, 1H, 3′OH), 4.19-4.02 (m, 2H, H2′),3.90-3.80 (m, 1H, H4′), 3.69 (s, 6H, 2×—OCH₃), 3.49 (t, 2H, H3′),2.93-2.74 (m, 2H, H5′).

¹³C NMR (DMSO-d₆): δδ 165.6, 157.9, 152.8, 151.6 (adenin CH), 150.2,144.6, 143.1 (adenin CH), 135.7, 135.4, 132.4 (Ar), 129.4 (Ar), 128.5(Ar), 128.4 (Ar), 127.7 (Ar), 127.5 (Ar), 126.4 (Ar), 125.2, 113.0 (Ar),85.0, 84.5 (1′C), 79.6 (4′C), 63.6 (5′C), 61.4 (2′C), 60.9 (3′C), 54.9(—OCH₃).

6-N-Benzoyl-2′-O-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′,3′-secoadenosine(103-A)

Nucleoside 102-A (2.01 g, 2.98 mmol) was coevaporated with an. MeCN(2×30 mL) and dried overnight. The resulting residue was dissolved inan. DCM (150 mL) along with an. DBU (900 mg, 5.96 mmol) and the mixturewas cooled to -70° C. 0.5 M benzoyl chloride solution (6.56 mL, 3.28mmol) was slowly added to the reaction mixture. The reaction mixture wasstirred for 1 h at −70° C. and subsequently allowed to reach rtwhereupon EtOH (5 mL was added). The reaction mixture was washed withsat. aq. NaHCO₃ (3×150 mL) and brine (150 mL). The combined aqueousphase was back-extracted with CH₂Cl₂ (100 mL). The organic phases werecombined and evaporated. The resulting residue was purified by columnchromatography (0-100% EtOAc in petroleum ether) affording the productnucleoside 103-A as a white foam after evaporation of the solvents.

Yield: 1.69 g (73%).

R_(f): 0.49 (EtOAc).

¹H NMR (DMSO-d₆): δ 11.28 (s, 1H, NH), 8.82 (s, 1H, adenine CH), 8.76(s, 1H, adenine CH), 8.06 (d, 2H, Ar), 7.79 (d, 2H, Ar), 7.55-7.40 (m,6H, Ar), 7.25-6.89 (m, 10, Ar), 6.77 (dd, 4H, Ar), 6.51 (t, 1H, H1′),4.99 (m, 2H, H2′), 4.91 (t, 1H, 3′OH), 3.89 (ap. s, 1H, H4′), 3.72 (s,6H, 2×—OCH₃), 3.54 (m, 2H, H3′), 2.81 (m, 2H, H5′).

¹³C NMR (DMSO-d₆): δ 157.9, 152.4, 150.5, 144.6, 142.9 (Adenine CH),135.6, 133.6 (Ar), 132.4 (Ar), 129.0 (Ar), 128.8 (Ar), 128.4 (Ar), 127.5(Ar), 125.2 (Ar), 113.0 (Ar), 85.1, 81.5 (C1′), 79.9 (C4′), 63.8 (C2′),63.5, 54.9 (—OCH₃).

ESI-HiRes (mNa⁺): m/z: 802.2848 calc.: 802.2847.

6-N-Benzoyl-2′-O-benzoyl-3′-O-(2-cyanoethoxy(diisopropylamino)phosphino)-5′-O-(4,4′-dimethoxytrityl)-2′,3′-secoadenosine(104-A)

Nucleoside 103-A (1.69 g, 2.17 mmol) was coevaporated with an. MeCN(2×20 mL) and dried for 12 h in vacuo. The residue was dissolved in 20%DIPEA in MeCN (40 mL) and the resulting mixture was stirred.2-Cyanoethyl-N,N-diisopropylchlorophosphoramidite[P(Cl)(OCH₂CH₂CN)(N(iPr)₂); 1.0 mL] was added to the reaction mixturewhich was stirred for 40 min. Additional2-Cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.2 mL) was added andthe resulting mixture was stirred for 3 hours. EtOH (5 mL) was added andthe mixture was washed with sat. aq. NaHCO₃ (3×50 mL) and the aqueousphase was back-extracted with DCM (50 mL). The organic phases werepooled and evaporated. The crude product was purified by columnchromatography (0-100% EtOAc in petroleum ether) to afford white foamafter evaporation of the solvents.

Yield: 1.52 g (71%).

R_(f): 0.75 (5% MeOH in DCM). ³¹P NMR (MeCN): δ 148.9.

ESI-HiRes (mNa⁺): m/z: 1002.3885 calc.: 1002.3926.

4-N-Acetyl-5′-O-(4,4′-dimethoxytrityl)-2′,3′-secocytidine (102-C)

4-N-Acetylcytidine (11.75 g, 41.18 mmol) was coevaporated with an. pyr(50 mL). The resulting residue was dissolved in an. pyr (160 mL). DMT-Cl(4,4′-dimethoxytritylchloride; 16.76 g, 49.42 mmol) was added as a solidmaterial and the resulting mixture was stirred for 2 hours at rt. Thereaction mixture was washed with sat. aq. NaHCO₃ (3×50 mL), and theorganic phase was evaporated to yield a white foam which was dried. Thisresidue was dissolved in dioxane (500 mL) and water (100 mL). NaIO₄(10.62 g, 49.5 mmol) was dissolved in water (100 mL) and added to thedissolved nucleoside. The mixture was stirred for 1 h during which timea white precipitate was formed. Additional 400 mL dioxane was added andstirring was continued for 15 min. The precipitate was filtered of andwashed with dioxane (200 mL). The filtrates were combined and NaBH₄(1720 mg, 45.5 mmol) was added, and stirring was continued for 30 min.To neutralize the reaction mixture, a buffer (10 mL, 1:1—AcOH:pyridine)was added until pH 7 was reached. The reaction mixture was evaporated to300 mL and extracted with EtOAc (150 mL). The organic phase was washedwith sat. aq. NaHCO₃ (3×200 mL) and evaporated. The residue was purifiedby column chromatography with a gradient of MeOH in EtOAc to give theproduct as a white solid material after evaporation of the solvents.

Yield: 17.24g (71%).

R_(f): 0.19 (5% MeOH in CHCl₃).

¹H NMR (DMSO-d₆): δ 10.94 (s, 1H, NH), 8.08 (d, 1H, J=7.32 Hz, CytidineCH), 7.31-7.12 (m, 12H, Ar/Cytidine CH), 6.85 (d, 4H, Ar), 5.96 (t, 1H,H1′), 5.13 (t, 1H, 2′OH), 4.74 (t, 1H, 3′OH), 3.73 (s, 6H, 2×—OCH₃),3.63 (m, 3H, H2′/H4′), 3.43 (m, 2, H3′), 3.00 (m, 2H, H5′), 2.13 (s, 3H,—CH₃).

¹³C NMR (DMSO-d₆): δ 170.9, 162.3, 158.0, 155.4, 146.1 (Cytidine C5/C6),144.6, 135.6, 129.6 (ar), 127.7 (ar), 126.6 (ar), 113.1 (ar), 95.4(Cytidine C5/C6), 85.5, 84.7 (C1′), 79.4 (C2′/C4′), 63.8 (C5′), 61.7(C2′/C4′), 60.5 (C3′), 55.0 (—OCH₃), 24.3 (—CH₃).

ESI-HiRes (mNa⁺): m/z: 612.2298 calc.: 612.2316.

4-N-Acetyl-2′-O-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′,3′-secocytidine(103-C)

Nucleoside 102-C (3.03 g, 5.14 mmol) was coevaporated with an. toluene(2×30 mL) and dried for 12 h in vacuo. The resulting residue wasdissolved in an. DCM (150 mL) along with an. DBU (1.5 mL, 10.3 mmol) andthe resulting mixture was cooled to -70° C. Benzoyl chloride (6.56 mL,5.65 mmol) was slowly added to the reaction mixture. The reactionmixture was stirred for 1 h at −70° C. and subsequently allowed to reachrt whereupon EtOH (4 mL) was added. The reaction mixture was washed withsat. aq. NaHCO₃ (2×150 mL). The organic phases were combined andevaporated. The resulting residue was purified by column chromatography(0-5% MeOH in CHCl₃) affording product nucleoside 103-C as a white foamafter evaporation of the solvents.

Yield: 2.08 g (64%).

R_(f): 0.24 (5% MeOH in CHCl₃).

¹H NMR (DMSO-d₆): δ 10.97 (s, 1H, NH), 8.25 (d, 1H, Cytidine CH), 7.91(d, 2H, Ar), 7.65 (ap. t, 1H, Ar) 7.32-7.12 (m, 12H, Ar/Cytidine CH),6.83 (d, 4H, Ar), 6.34 (t, 1H, H1′), 4.84 (t, 1H, 3′OH), 4.58 (dq, 2H,H2′), 3.74 (s, 6H, 2×—OCH₃), 3.70-3.64 (m, 1H, H4′), 3.48 (m, 2H, H3′),3.07 (m, 2H, H5′), 2.14 (s, 3H, —CH₃).

¹³C NMR (DMSO-d₆): δ 171.0, 165.0, 162.5, 157.1, 145.5 (Cytidine C5/C6),144.6, 135.48, 133.6, 129.6 (Ar), 128.8 (Ar), 127.7 (Ar), 127.6 (Ar),126.6 (Ar), 113.1 (Ar), 95.8 (Cytidine C5/C6), 85.6, 82.0 (C1′), 79.6(C4′), 79.2 63.9 (C2′), 63.7, 60.5 (C3′), 54.9 (—OCH₃), 24.3 (—CH₃).

ESI-HiRes (mNa⁺): m/z: 716.2589 calc.: 716.2759.

4-N-Acetyl-2′-O-benzoyl-3′-O-(2-cyanoethoxy(diisopropylamino)phosphino)-5′-O-(4,4′-dimethoxytrityl)-2′,3′-secocvtidine(104-C)

Nucleoside 103-C (1.49 g, 2.15 mmol) was coevaporated with an. MeCN(2×20 mL). The residue was dissolved in 20% DIPEA in MeCN (20 mL) andthe mixture was stirred.2-Cyanoethyl-N,N-diisopropylchlorophosphoramidite[P(Cl)(OCH₂CH₂CN)(N(iPr)₂); 0.8 mL] was added to the mixture andstirring was continued for 40 min. Additional2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.4 mL) was added andstirring was continued for 3 h. EtOH (5 mL) was added and the resultingmixture was washed with sat. aq. NaHCO₃ (3×50 mL) and the aqueous phasewas back-extracted with DCM (50 mL). The organic phases were pooled andevaporated. The residue was purified by column chromatography (0-100%EtOAc inpetroleum ether) to afford nucleoside 104-C as a white foamafter evaporation of the solvents.

Yield: 940 mg (44%).

R_(f): 0.42 (5% MeOH in DCM).

³¹P NMR (MeCN): δ 148.8.

ESI-HiRes (mNa⁺): m/z: 916.3622 calc.: 916.3657.

5′-O-(4,4′-Dimethoxytrityl)-2-N-isobutyryl-2′,3′-secoguanosine (102-G)

2-N-Isobutyrylguanosine (11.68 g, 17.8 mmol) was coevaporated with an.pyr (50 mL). The resulting residue was dissolved in an. pyr (100 mL).DMT-Cl (4,4′-dimethoxytritylchloride; 7.26 g, 21.46 mmol) was added as asolid material and the reaction mixture was stirred for 1 h at rt. DMAP(50 mg, 0.40 mmol) was added and the resulting mixture was stirred foradditional 12h. The reaction mixture was then washed with sat. aq.NaHCO₃ (3×50 mL) and the organic phase evaporated to yield a white foam.This residue was dissolved in dioxane (250 mL) and water (50 mL). NaIO₄(4.57 g, 21.3 mmol) was dissolved in water (50 mL) and was added to thedissolved nucleoside. The mixture was stirred for 1 h during which timea white precipitate was formed. Additional 200 mL dioxane was added andstirring was continued for 15 min. The precipitate was filtered of andwashed with dioxane (100 mL). The filtrates were collected and NaBH₄(748 mg, 19.77 mmol) was added and the resulting mixture was stirred for30 min at rt. To neutralize a buffer (10 mL, 1:1—AcOH:pyridine) wasadded until pH 7 was reached. The volume of the resulting mixture wasreduced to 150 mL and extraction was performed using EtOAc (150 mL). Theorganic phase was washed with sat. aq. NaHCO₃ (3×100 mL) and evaporated,and the residue was purified by column chromatography using a gradientof 0-10% (1:1 MeOH:i-PrOH) in DCM to yield the product as a white solidmaterial after evaporation of the solvents.

Yield: 8.02g (68%).

R_(f): 0.24 (7% MeOH in CH₂Cl₂).

¹³C NMR (DMSO-d₆): δ 180.2, 157.9, 154.9, 147.8, 144.7, 135.4, 129.3(Ar), 127.5 (Ar), 127.4 (Ar), 126.4 (Ar), 120.4, 113.0 (Ar), 85.2, 85.1(C1′), 79.9 (C4′), 63.5 (C5′), 61.7 (C2′), 61.1 (C3′), 54.9 (—OCH₃),34.7 (quaternary i-Pr), 18.9 (i-Pr), 18.8 (i-Pr).

MALDI-HiRes (mNa⁺): m/z: 680.2679 calc.: 680.2691.

2′-O-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-2-N-isobutyryl-2′,3′-secoguanosine(103-G)

Nucleoside 102-G was suspended in an. toluene (2×30 mL) and evaporated.The resulting residue was dried for 12 h in vacuo. The residue wasdissolved in an. DCM (100 mL) along with an. DBU (0.9 mL, 6.1 mmol) andthe resulting mixture was cooled to -70° C. Benzoyl chloride (390 μL,3.36 mmol) was slowly added to the reaction mixture. The reactionmixture was stirred for 1 h at −70° C. and subsequently allowed to reachrt whereupon EtOH (4 mL) was added. The resulting mixture was washedwith sat. aq. NaHCO₃ (2×100 mL), and the organic phases were combinedand evaporated. The resulting residue was purified by columnchromatography (0-5% MeOH in CHCl₃) affording nucleoside 103-G as awhite foam after evaporation of the solvents.

Yield: 1.49 g (63%).

R_(f): 0.47 (7% MeOH in CH₂Cl₂).

¹H NMR (DMSO-d₆): δ 12.10 (s, 1H, NH), 11.72 (s, 1H, NH), 8.32 (s, 1H,guanidine H8), 7.85-7.79 (m, 2H, Ar), 7.65-7.63 (m, 1H, Ar), 7.51-7.45(m, 2H, Ar), 7.26-6.97 (m, 11H, Ar), 6.79 (m, 4H, Ar), 6.18 (t, 1H,H1′), 5.04-4.82 (m, 3H, H2′/3′OH), 3.82 (m, 1H, H4′), 3.72 (s, 6H,2×—OCH₃), 3.49 (t, 2H, H3′), 3.03-2.74 (m, 3H, H5′/quaternary i-Pr),1.11 (ap. t, 6H, 2×—CH₃).

¹³C NMR (DMSO-d₆): δ 180.1, 164.9, 157.8, 154.8, 148.6, 147.9, 144.6,138.4, 135.5, 135.3, 133.6, 129.3 (Ar), 129.0 (ar), 128.8 (ar), 128.7(ar), 127.6 (ar), 127.4 (ar), 126.3 (ar), 120.6, 112.9 (ar), 85.1, 82.0(C1′), 80.1 (C4′), 63.7, 63.3 (C5′), 61.0 (C3′), 54.8 (—OCH₃), 34.6(quaternary i-Pr), 18.8 (—CH₃), 18.6 (—CH₃).

ESI-HiRes (mNa⁺): m/z: 784.2943 calc.: 784.2953.

2′-O-Benzoyl-3′-O-(2-cyanoethoxy(diisopropylamino)phosphino)-5′-O-(4,4′-dimethoxytrityl)-2-N-isobutyryl-2′,3′-secoguanosine(104-G)

Nucleoside 103-G (1.45 g, 1.9 mmol) was coevaporated with an. MeCN (2×20mL). The residue was dissolved in 20% DIPEA in MeCN (20 mL) and theresulting mixture was stirred.2-Cyanoethyl-N,N-diisopropylchlorophosphoramidite[P(Cl)(OCH₂CH₂CN)(N(iPr)₂); 0.65 mL] was added to the reaction mixtureand stirring was continued for 40 min. EtOH (5 mL) was added and theresulting mixture was washed with sat. aq. NaHCO₃ (3×50 mL), and theaqueous phase was back-extracted with DCM (50 mL). The organic phaseswere pooled and evaporated. The residue was precipitated from petroleumether from a solution in EtOAc to furnish amidite 104-G as a white solidmaterial after drying.

Yield: 607 mg (33%).

R_(f): 0.3 (1:3 Acetone:toluene).

³¹P NMR (MeCN): δ 148.6.

ESI-HiRes (mNa⁺): m/z: 984.4028 calc.: 984.4031.

Example 12 Synthesis of Piperazino-functionalised Monomeric BuildingBlocks

The example describes procedures that have been conducted in order toexemplify synthesis of monomeric amidite building blocks having an aminofunctionality attached at the C2′-position of a monomer, i.e. synthesisof the C2′-piperazino-functionalised monomeric building block 111(Amidite J; base=uracil) starting from nucleoside 103-U via compounds105, 106, 107, 108, 109 and 110.

2′-O-Benzoyl-3′-O-tert-butyldimethelsilyl-5′-O-(4,4′-dimethoxytrityl)-2′,3′-secouridine(105)

Nucleoside 103-U (328 mg, 0.50 mmol) was dissolved in an. pyridine (2mL) and stirred at rt. TBDMSCI (113 mg, 0.75 mmol) was added to thereaction mixture that was stirred for 19 h. Water (1 mL) was then addedand stirring was continued for additional 15 min whereupon the reactionmixture was diluted with DCM (50 mL) and washed with sat. aq. NaHCO₃(2×25 mL) and brine (25 mL). The organic phase was dried over Na₂SO₄,filtered and evaporated to dryness under reduced pressure. The residuewas purified by silica gel column chromatography using MeOH (0-8%) inDCM as eluent thus affording nucleoside 105 as a white solid material.Yield 294 mg (78%). ¹H NMR (300 MHz, DMSO-d₆) δ 11.46 (s, 1H, NH),7.95-7.79 (m, 3H), 7.71-7.63 (m, 1H), 7.57-7.48 (m, 2H), 7.37-7.13 (m,9H), 6.84 (d, J=8.8 Hz, 4H), 6.22 (t, J=5.7 Hz, 1H, H1′), 5.58 (d, J=8.0Hz, 1H, H5), 4.69-4.45 (m, 2H, H2′), 3.80-3.64 (m, 8H, 2×OMe and H4′),3.54 (t, J=4.7 Hz, 1H, H3′), 3.09-2.97 (m, 2H, H5′), 0.73 (s, 9H, 3×Me),−0.07 and −0.09 (2×s, 6H, 2×Me). ¹³C NMR (75.5 MHz, DMSO-d₆): δ 165.0,163.1, 158.0, 151.1, 144.7, 140.6, 135.5, 135.4, 133.7, 129.6, 129.1,129.0, 128.8, 127.8, 127.6, 126.6, 113.1, 102.3, 85.5, 81.1, 79.2, 63.4,63.1, 62.1, 55.0, 25.6, 17.7, 2×-5.7. ESI-HRMS: m/z 789.3147 ([M+Na]⁺,C₄₃H_(SO)N₂O₉Si.Na calc. 789.3178).

3′-O-tert-Butyldimethelsilyl-5′-O-(4,4′-dimethoxytrityl)-2′,3′-secouridine(106)

NaOH (845 mg, 21.1 mmol) was mixed with an. MeOH (200 mL) and theresulting mixture was cooled to 0° C. Nucleoside 105 (3.10 g, 4.05 mmol)was dissolved in an. MeOH (40 mL) and the resulting mixture was added tothe above mixture and the resulting reaction mixture was stirred for 2.5h. Sat. aq. NH₄Cl (10 mL) was added and stirring was continued foradditional 10 min whereupon water (100 mL) was added and extraction wasperformed using DCM (4×200 mL). The organic phase was evaporated todryness under reduced pressure and the residue then co-evaporated withan. pyridine (10 mL). The residue was purified by silica gel columnchromatography using MeOH (5-10%) in DCM as eluent thus affordingnucleoside 106 as a white solid material. Yield 2.54 g (95%). ¹H NMR(300 MHz, DMSO-d₆): δ 11.35 (s, 1H, NH), 7.66 (d, J=7.6 Hz, 1H, H6),7.33-7.14 (m, 9H), 6.88-6.82 (m, 4H), 5.82 (t, J=6.0 Hz, 1H, H1′), 5.53(d, J=8 Hz, 1H, H5), 5.11 (t, J=5.8 Hz, 1H, 2′-OH), 3.73 (s, 6H, 2×OMe),3.69-3.45 (m, 5H, H2′, H4′ and H3′), 3.01-2.93 (m, 2H, H5′), 0.76 (s,9H, 3×Me), −0.03 and −0.05 (2×s, 6H, 2×Me). ¹³C NMR (75.5 MHz, DMSO-d₆):δ 163.2, 158.0, 151.6, 144.8, 135.6, 135.4, 129.6, 127.7, 127.6, 126.6,113.1, 101.8, 85.4, 83.5, 78.5, 63.3, 61.8, 61.0, 55.0, 25.6, 17.7,2×-5.6. ESI-HRMS: m/z 685.2885 ([M+Na]⁺, C₃₆H₄₆N₂O₈Si.Na calc.685.2916).

3′-O-tert-Butyldimethylsilyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-methanesulfonyl-2′,3′-secouridine(107)

Nucleoside 106 (927 mg, 1.40 mmol) was dissolved in an. pyridine (20 mL)and the resulting mixture was stirred at 0° C. MsCl (220 μL, 2.83 mmol)was added dropwise and the resulting mixture was stirred for 3 h at rt.EtOH (2 mL) was added and stirring was continued for additional 10 min.The mixture was then evaporated to dryness and the residue was purifiedby silica gel column chromatography using MeOH (0-7%) in DCM as eluentthus affording nucleoside 107 as a white foam. Yield 834 mg (81%). ¹HNMR (300 MHz, DMSO-d₆): δ 11.49 (s, 1H, NH), 7.77 (d, J=8.2 Hz, 1H, H6),7.35-7.14 (m, 9H), 6.86 (d, J=8.5 Hz, 4H), 6.11 (t, J=5.7 Hz, 1H, H1′),5.60 (d, J=8.1 Hz, 1H, H5), 4.49 (d, J=5.5 Hz, 2H, H2′), 3.77-3.48 (m,9H, 2×OMe, H4′ and H3′), 3.22 (s, 3H, Me), 3.10-2.89 (m, 2H, H5′), 0.77(s, 9H, 3×Me), −0.02 and −0.04 (2×s, 6H, 2×Me). ¹³C NMR (75.5 MHz,DMSO-d₆): δ 163.1, 158.0, 151.7, 144.7, 140.5, 135.5, 135.3, 129.6,127.8, 127.6, 126.6, 113,1, 102.4, 85.5, 80.6, 79.1, 67.8, 63.1, 61.8,55.0, 36.8, 25.6, 17.7, −5.6, −5.7. ESI-HRMS: m/z 763.2662 ([M+Na]⁺,C₃₇H₄₈N₂O₁₀SSi.Na calc. 763.2692).

3′-O-tert-butyldimethylsilyl-2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-2′-piperazino-2′,3′-secouridine(108)

Nucleoside 107 (276 mg, 0.373 mmol) was dissolved in an. pyridine (3 mL)and piperazine (3.21 g, 37.3 mmol) was added under stirring at rt. Thereaction mixture was then heated to 150° C. and stirred for 1 h followedby cooling to rt. The reaction mixture was diluted with sat. aq. NaHCO₃(200 mL) whereupon extraction was performed using chloroform (7×100 mL).The organic phase was dried over Na₂SO₄, filtered and evaporated todryness. The residue was purified by silica gel column chromatographyusing first MeOH (0-5%) in DCM and then half sat. methanolic ammonia(5%) in DCM as eluent systems thus affording nucleoside 108 as a whitesolid material. Yield 182 mg (67%). ¹H NMR (300 MHz, DMSO-d₆): δ 7.64(d, J=8.1 Hz, 1H, H6), 7.34-7.13 (m, 9H), 6.85 (d, J=7.8 Hz, 4H), 5.98(t, J=6.0 Hz, 1H, H1′), 5.53 (d, J=8.1 Hz, 1H, H5), 3.72 (s, 6H, 2×OMe),3.68-3.51 (m, 3H, H4′ and H3′), 3.04-2.90 (m, 2H, H5′), 2.77-2.54 (m,6H, H2′, piperazino), 2.48-2.27 (m, 4H, piperazino), 0.77, (s, 9H,3×Me), −0.02 and −0.04 (2×s, 6H, 2×Me). ¹³C NMR (75.5 MHz, DMSO-d₆): δ163.2, 158.0, 151,3, 144.8, 141.1(C6), 135.6, 135.4, 129.5, 127.7,127.6, 126.6, 113.1, 113.1, 101.8 (C5), 85.4, 81.3 (C1′), 78.3, 63.1,62.1, 60.1, 55.0 (OMe), 55.0(OMe), 53.8, 45.3, 25.7 (Me), 17.8,-5.5(Me), −5.6 (Me). ESI-HRMS: m/z 731.3859 ([M+H]⁺, C₄₀H₅₄N₄O₇Si.H calc.731.3834).

3′-O-tert-Butyldimethylsilyl-2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-2′-(4-N-trifluoroacetyl)piperazino-2′,3′-secouridine(109)

Nucleoside 108 (102 mg, 0.14 mmol) was dissolved in an. MeOH (2 mL) andthe resulting mixture was stirred at rt. DMAP (10 mg, 0.08 mmol) andethyl trifluoroacetate (22 μL, 0.184 mmol) were added and stirring wascontinued for 16 h. The mixture was then evaporated to dryness underreduced pressure and the residue was purified by silica gel columnchromatography using MeOH (0-2%) in DCM as eluent thus affordingnucleoside 109 as a white solid material. Yield 100 mg (86%). ¹H NMR(300 MHz, DMSO-d₆): δ 11.37 (s, 1H, NH), 7.66 (d, J=7.8 Hz, 1H, H6),7.38-7.12 (m, 9H), 6.93-6.80 (m, 4H), 6.00 (t, J=5.9 Hz, 1H, H1′), 5.54(d, J=7.8 Hz, 1H, H5), 3.73 (s, 6H, 2×OMe), 3.70-3.40 (m, 7H, H3′, H4′and piperazino), 3.08-2.92 (m, 2H, H5′), 2.90-2.52 (m, 6H, H2′ andpiperazino), 0.77 (s, 9H, 3×Me), 0.00 and -0.04 (2×s, 6H, 2×Me). ¹³C NMR(75.5 MHz, DMSO-d₆): δ 163.2, 158.0, 151.3, 144.8, 140.8, 135.6, 135.4,129.5, 127.7, 127.6, 126.6, 113.1, 102.0, 85.5, 81.0, 78.2, 63.1, 62.0,58.9, 55.0, 52.6, 52.0, 45.4, 43.0, 25.6, 17.8,-5.6,-5.6. ESI-HRMS: m/z849.3452 ([M+Na]⁺, C₄₂H₅₃F₃N₄O₈Si.Na calc. 849.3477).

2′-Deoxy-5′-O-(4,4′-dimethoxytrityl)-2′-(N-trifluoroacetyl)piperazino-2′,3′-secouridine(110)

Nucleoside 109 (251 mg, 0.304 mmol) was co-evaporated with an. THF (2×5mL) and then dissolved in an. THF (10 mL). 1M TBAF in THF (606 μL, 0.606mmol) was added dropwise under stirring to this mixture at rt during 1 hand stirring at rt was then continued for 21 h. The reaction mixture wasevaporated to dryness under reduced pressure and the residue thendissolved in EtOAc (40 mL). The resulting mixture was washed with sat.aq. NaHCO₃ (3×10 mL) and water (3×10 mL), and the separated organicphase was dried over Na₂SO₄, filtered and evaporated to dryness underreduced pressure. The residue was purified by silica gel columnchromatography using EtOAc (60-100%) in petroleum ether as eluent thusaffording nucleoside 110 as a white solid material. Yield 111 mg (51%).¹H NMR (300 MHz, DMSO⁻d₆): δ 11.36 (s, 1H, NH), 7.66 (d, J=8.1 Hz, 1H,H6), 7.34-7.27 (m, 4H), 7.24-7.14 (m, 5H), 6.92-6.82 (m, 4H), 5.98 (t,J=6.1 Hz, 1H, H1′), 5.53 (d, J=7.3 Hz, 1H, H5), 4.80 (t, J=5.3 Hz, 1H,3′-OH), 3.73 (s, 6H, 2×OMe), 3.63-3.38 (m, 8H, H4′, H3′ and piperazino),3.07-2.89 (m, 2H, H5′), 2.77 (t, J=5.8 Hz, 2H, H2′), 2.66-2.54 (m, 3H,piperazino). ¹³C NMR (75.5 MHz, DMSO-d₆): δ 163.2, 158.0, 151.2, 144.8,140.9, 135.6, 135.5, 129.6, 129.6, 127.8, 127.6, 126.6, 113.1, 101.9,85.4, 81.3, 79.2, 63.5, 60.7, 59.1, 55.0, 52.7, 52.1, 45.4, 42.9.ESI-HRMS: m/z 735.2585 ([M+Na]⁺, C₃₆H₃₉F₃N₄O₈.Na calc. 735.2612).

3′-(2-Cyanoethoxy(diisopropylamino)phosphino)-2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-2′-(N-trifluoroacetyl)piperazino-2′,3′-secouridine(111)

Nucleoside 110 (91 mg, 0.128 mmol) was co-evaporated with an. DCM (2×5mL) and then dissolved in an. DCM (2.5 mL). DIPEA (111 μL, 0.64 mol) wasadded under stirring to this mixture at rt whereupon 2-cyanoethylN,N-diisopropylphosphoramidochloridite (57 μL, 0.256 mmol) was addeddropwise. Stirring at rt was continued for 1 h, whereupon EtOH (0.5 mL)was added followed by stirring for additional 10 min. DCM (40 mL) wasadded followed by washing with sat. aq. NaHCO₃ (20 mL). The separatedorganic phase was dried over Na₂SO₄, filtered and evaporated to drynessunder reduced pressure. The residue was purified by silica gel columnchromatography using EtOAc (60-80%) in petroleum ether as eluent thusaffording the amidite 111 as a white solid material. Yield 106 mg (91%).³¹P NMR (CDCl₃) δ 150.0 and 149.5. ESI-HRMS: m/z 913.3841 ([M+H]⁺,C₄₅H₅₆F₃N₆O₉P.H calc. 913.3871).

Example 13 Synthesis of Oligonucleotides ContainingPiperazino-functionalised Monomeric Building Blocks

By using methods described in Example 1, efficient incorporation ofmonomer J with a free terminal NH in the piperazino moiety wasaccomplished using RNA amidites and amidite 111. The coupling yields ofthis amidite were above 95%.

The invention claimed is:
 1. An oligomer comprising one or more2′-3′-seco-nucleomonomers and one or more natural or non-naturalnucleotide monomers, wherein the oligomer is a single stranded RNAoligomer and the 2′-3′-seco-nucleomonomers are monomer D

wherein Base is a nucleobase.
 2. The oligomer of claim 1, wherein theoligomer is from 8 to 62 monomers in length.
 3. The oligomer of claim 1,comprising from one to five 2′-3′-seco-nucleomonomers.
 4. The oligomerof claim 1, wherein one or more of the nucleotide monomers is a2′-O-alkyl-RNA nucleotide analogue.
 5. The oligomer of claim 1, whereinone or more of the monomers are linked by a phosphorothioate linkage ora boranophosphate linkage.
 6. The oligomer of claim 1, wherein theoligomer is from 14 to 26 monomers in length.
 7. The oligomer of claim1, comprising a monomer nucleobase sequence that is complementary to atarget nucleotide sequence.
 8. The oligomer of claim 1, wherein theoligomer has increased or prolonged stability towards enzymaticdegradation in a cell as compared to an oligonucleotide having the samenucleobase sequence, wherein the oligonucleotide is composed of onlynatural RNA monomers.
 9. The oligomer of claim 1, wherein the oligomercomprises two, three, four, or five 2′-3′-seco-nucleomonomers.